Biodegradable biopolymers, method for their preparation and functional materials constituted by these biopolymers

ABSTRACT

A biodegradable biopolymer material consists of silk fibroin from domesticated silkworm; silk fibroin from wild silkworm; a composite material comprising silk fibroin from domesticated silkworm and silk fibroin from wild silkworm; or a composite material comprising either silk fibroin from domesticated silkworm or silk fibroin from wild silkworm and at least one secondary substance selected from the group consisting of cellulose, chitin, chitosan, chitosan derivatives, keratin from wool and polyvinyl alcohol. The material may be prepared by, for instance, casting an aqueous solution of domesticated silkworm silk fibroin on the surface of a substrate and then cast drying the applied solution. The biodegradable biopolymer material is effectively used as, for instance, a metal ion-adsorbing material, a sustained release substrate for a useful substance such as a medicine, a biological cell-growth substrate and a biodegradable water-absorbing material.

CROSS REFERENCE TO RELATED APPLICATIONS

This nonprovisional application is a continuation application whichclaims the benefit of pending U.S. patent application Ser. No.10/458,277, filed Jun. 11, 2003, now abandoned, and claims priority ofApplication Number 2002-178126, filed Jun. 19, 2002, in Japan. Thedisclosures of the prior applications are hereby incorporated herein intheir entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a biodegradable biopolymer material,which is degraded while being decomposed by the action of an enzyme. Thebiodegradable biopolymer material is thus converted into smallmolecules. The present invention also relates to a method for thepreparation of the biodegradable biopolymer material, as well as afunctional material containing the material, such as a metalion-adsorbing material, a sustained release carrier for a usefulsubstance, a biological cell-growth substrate and a biodegradablewater-absorbing material.

2. Description of the Related Art

Materials consisting of organic polymers and possessing biodegradabilityare well known in the marketplace. In the medical field, materials havefrequently been used which are biologically decomposed and degradedthrough the action of an enzyme to form small molecules. Materialsrecently put into practical use include, as typical examples,poly(oxy-acids), such as poly(lactic acid) and poly(glycolic acid) amongthe biodegradable organic polymers. These materials have widely beenused as implanting materials to be embedded in the living bodies,materials for in vivo delivery or carriers for sustained release ofmedicines.

Poly(lactic acid) and poly(glycolic acid) show excellent resistance tochemicals. Poly(lactic acid) and poly(glycolic acid) are non-toxic andsusceptible to hydrolysis. Accordingly, they have widely been used asmaterials capable of being decomposed and absorbed in vivo. Moreover,poly(glycolic acid) can be prepared as a very high molecular weightpolymer and therefore, it is useful as a material, which should haveexcellent mechanical or dynamic characteristic properties, such as hightensile strength. Poly(lactic acid) and poly(glycolic acid) have beenused as, for instance, biodegradable and bioabsorbable suture.

Moreover, in the medical field, silk sutures from domesticated silkwormhave been used for surgical operations. The use of the silk fiber fromdomesticated silkworm as sutures for surgical operations dates back tothe beginning of the eleventh century. The total volume of the suturestraded in this country is equivalent to about six billion yen a year (in1985), 46% of which corresponds to the volume of the silk sutures. Thesilk fiber is excellent in, for instance, tensile strength and knotstrength and can easily be sterilized. For this reason, silk fiber hasfavorably been used for sutures. Even when judging from the actualconditions of the use of the conventional silk sutures, the silk fibercan be sterilized easily. It never biologically decomposes in a shortperiod of time when embedded in the living body. Further, when it isimplanted in the living body, it only causes an insignificantantigen-antibody reaction, if any, with the biological tissues.

The cocoon fiber (the silk fiber) is a protein fiber produced and spunby matured larvae of silkworm. The silkworms are divided into twogroups, domesticated silkworms reared in farmhouses and wild typesilkworms. Silk fibroin fibers are those obtained by removing sericin asan adhesive substance, which covers the surface of the cocoon fiber, bytreating the cocoon fiber with, for instance, an alkali.

The silk fibers from wild silkworm in general mean those produced andspun by, for instance, Antheraea pernyi, Antheraea yamamai, Antheraeamilitta, Antheraea assama, Philosamia cynthia ricini and Philosamiacynthia pryeri.

The foregoing silk suture is a non-absorbent material, which is neverdecomposed within a short period of time. Accordingly, it would remainin the living body after the suture. For this reason, it has been usedfor purposes different from those of the threads for a suture made frompoly(oxy-acids), such as poly(lactic acid) and poly(glycolic acid),which are absorbed in the body and are decomposed into water and carbondioxide within several weeks after the suture.

With respect to the foregoing metal ion-adsorbing material and sustainedrelease carrier for useful substances consisting of the aforementionedbiodegradable biopolymer, no product having satisfactory characteristicproperties has been proposed yet.

As discussed above, poly(lactic acid) has been widely used as abiodegradable and bioabsorbable material. However, it suffers from theproblems that the production cost thereof is too high, it is tooexpensive, it has high crystallizability, it is too hard, and it isinferior in the compatibility with soft tissues. Moreover, it alsosuffers from problems that its rate of decomposition cannot be easilycontrolled and that control of its biodegradability is also difficult,even if this material is chemically modified.

Further, fibrous poly(lactic acid) has a glass transition temperaturesimilar to that of polyethylene terephthalate fiber. Accordingly, thepoly(lactic acid) fibers possess mechanical properties similar to thoseobserved for the polyethylene terephthalate fibers. However, poly(lacticacid) or the like has a crystallization velocity slower than thatobserved for polyethylene terephthalate and fibers of poly(lactic acid)are not sufficiently oriented or satisfactorily crystallized even whenthey are passed through the usual spinning and/or orientation steps. Forthis reason, additional problems arise when putting them into practicaluse. For instance, the tensile strength and dimensional stability ofpoly(lactic acid) are insufficient.

In addition, the higher the molecular weight of the foregoingpoly(oxy-acids), the slower the rate of the decomposition thereof. Thus,it is necessary to produce poly(lactic acid) and poly(glycolic acid)whose molecular weight is controlled to control the decomposition speedof these polymers. However, the production of such polymers requires alot of labor and the use of highly advanced techniques which require agreat deal of skill. For this reason, the use of poly(oxy-acids) hasbeen limited to medical applications, such as absorbent sutures andcosmetic applications. Accordingly, there has been a strong desire for aproduction process, which is inexpensive or economical, and does notrequire any skilled technique.

As discussed above, a suture of silk differs from sutures ofpoly(oxy-acids), such as poly(lactic acid) and poly(glycolic acid),which are decomposed into water and carbon dioxide in vivo. Accordingly,there is a strong desire for the development of a biodegradable materialwhose biodegradability in vivo can be controlled, which does not sufferfrom any problem concerning the biological safety, whose production costis very low, which can biologically be decomposed without producing anycytotoxic products, which does not form any harmful substance such asformaldehyde as a by-product, and which is safe to the biologicaltissues.

The silk protein, which can be used as a raw material for the foregoingsilk suture, is a naturally occurring polymer material produced throughthe biosynthesis of silkworms, which is excellent in the biologicalcompatibility with the biological tissues and has good moldingproperties. Therefore, if by-products of silk, obtained in the processfor preparing raw silk and silk products, are used as starting materialfor the sutures, one can save the cost of raw materials. Moreover, silkproteins include a large number of active sites rich in chemicalreactivity. Therefore, the fields of application (such as the use asmedical materials) can be widely extended if a technique, which permitsthe control of the biodegradability or biochemical properties of silkfibroin, can be developed, for instance, through hybrid processingsand/or chemical modification. For this reason, there has been a strongdesire for the development of a novel biodegradable material, which canbe used effectively in the medical field, and which uses biopolymersfrom insects as starting materials and secondary substances capable ofbeing combined (hybrid or hybridized with the former compositematerials).

SUMMARY OF THE INVENTION

Accordingly, it is generally an object of the present invention to solvethe problems associated with the foregoing conventional techniques. Morespecifically, it is an object of the present invention to provide abiodegradable biopolymer material consisting of a silk protein excellentas a polymeric substrate; a hybridized biodegradable biopolymer materialcomprising the silk protein and a specific secondary substancehybridized together and having unique characteristic properties notobserved for the silk protein alone; a method for the preparation of thesame; and functional materials consisting of the foregoing biodegradablebiopolymer materials, such as a metal ion-adsorbing material, asustained release carrier for a useful substance, a biologicalcell-growth substrate, and a biodegradable and water absorbablematerial.

The silk fibers from domesticated silkworm and wild silkworm are fibrousmaterials produced and spun by silkworm. They have strong resistance tochemicals, for instance, chemical agents and enzymes, since they havefibrous structures, as determined by the X-ray diffraction analysis.This is why the silk fiber from domesticated silkworm is classified asbiologically non-absorbent material. The inventors of this inventionhave conducted various studies to provide a material comprising such asilk protein having good biodegradability, while making use of theexcellent biochemical properties of the silk protein. The inventors alsodeveloped a technique for preparing a novel material whosebiodegradability can be controlled by using silk fibroin fromdomesticated silkworm as a starting material and combining the startingmaterial with a specific secondary substance. The inventors have furtherinspected for degradation behavior observed for a novel compositematerial obtained during the development process when acting an enzymeon the composite material. The inventors have found that a biopolymermaterial possessing biodegradability can be provided and have thuscompleted the present invention.

The biodegradable biopolymer material of the present invention ischaracterized in that it consists of silk fibroin from domesticatedsilkworm; silk fibroin from wild silkworm; a composite materialcomprising silk fibroin from domesticated silkworm and silk fibroin fromwild silkworm; or a composite material comprising either silk fibroinfrom domesticated silkworm or silk fibroin from wild silkworm and atleast one secondary substance selected from the group consisting ofcellulose, chitin, chitosan, chitosan derivatives, keratin from wool andpolyvinyl alcohol.

In this respect, the biodegradable biopolymer material may be capable ofbeing biologically degraded by the action of at least one enzymeselected from the group consisting of proteases, collagenases andchymotrypsin.

The shape of the biodegradable biopolymer material may be fibrous,membrane-like, powdery, gel-like or porous.

The method for the preparation of a biodegradable biopolymer materialaccording to the present invention comprises the steps of applying, ontothe surface of a substrate, an aqueous solution of silk fibroin fromdomesticated silkworm, an aqueous solution of silk fibroin from wildsilkworm, an aqueous mixed solution containing an aqueous solution ofsilk fibroin from domesticated silkworm and an aqueous solution of silkfibroin from wild silkworm or an aqueous mixed solution comprisingeither an aqueous solution of silk fibroin from domesticated silkworm oran aqueous solution of silk fibroin from wild silkworm and an aqueoussolution of at least one secondary substance selected from the groupconsisting of cellulose, chitin, chitosan, chitosan derivatives, keratinfrom wool and polyvinyl alcohol; and then drying the applied solution todryness to form a film-like biodegradable biopolymer material, whereinif using the aqueous mixed solution, the aqueous solutions as theconstituents of the aqueous mixed solution are uniformly admixedtogether by stirring them such that they do not undergo any gelation,precipitation and/or coagulation reaction to thus prepare the aqueousmixed solution.

Moreover, a powdery biodegradable biopolymer material of the presentinvention can be prepared by freezing the foregoing aqueous solution ofsilk fibroin from domesticated silkworm, the foregoing aqueous solutionof silk fibroin from wild silkworm or the foregoing aqueous mixedsolution, and then drying the frozen aqueous solution under a reducedpressure. The mixed aqueous solution is prepared by the mixing methodused above. Further, a gel-like biodegradable biopolymer material of thepresent invention can be prepared by adjusting the pH value of theforegoing aqueous solution of silk fibroin from domesticated silkworm,the foregoing aqueous solution of silk fibroin from wild silkworm or theforegoing aqueous mixed solution to a level falling within the acidicregion, and then coagulating the entire aqueous solution to give agel-like biodegradable biopolymer material. Incidentally, a poroussubstance can be prepared by subjecting the gel-like product of thebiodegradable biopolymer material to lyophilization.

In the preparation of the foregoing aqueous mixed solution, theconcentrations of the aqueous solution of silk fibroin from domesticatedsilkworm, the aqueous solution of silk fibroin from wild silkworm, andthe aqueous solution of the secondary substance preferably range from0.1 to 5% w/v, respectively. If the concentration is less than 0.1% w/v,the amount of the aqueous solutions required for the preparation of thecomposite material increases. This is not preferred for operationefficiency. If the concentration exceeds 5% w/v, it is difficult touniformly admix two solutions and, as a result, it is impossible toprepare any composite material having uniform quality.

The metal ion-adsorbing material according to the present inventionconsists of a biodegradable biopolymer material, which is silk fibroinfrom domesticated silkworm; silk fibroin from wild silkworm; a compositematerial comprising silk fibroin from domesticated silkworm; silkfibroin from wild silkworm; or a composite material comprising eithersilk fibroin from domesticated silkworm or silk fibroin from wildsilkworm and at least one secondary substance selected from the groupconsisting of cellulose, chitin, chitosan, chitosan derivatives, keratinfrom wool and polyvinyl alcohol. The metal ions may be anti-bacterialmetal ions, such as silver, copper and cobalt ions or metal ions presentin waste water.

The sustained release carrier for a useful substance according to thepresent invention consists of a biodegradable biopolymer material, whichis silk fibroin from domesticated silkworm; silk fibroin from wildsilkworm; a composite material comprising silk fibroin from domesticatedsilkworm and silk fibroin from wild silkworm; or a composite materialcomprising either silk fibroin from domesticated silkworm or silkfibroin from wild silkworm and at least one secondary substance selectedfrom the group consisting of cellulose, chitin, chitosan, chitosanderivatives, keratin from wool and polyvinyl alcohol. It is alsocharacterized in that it can gradually release the useful substancesupported on the biodegradable biopolymer material while beingbiodegraded by the action of a protease, chymotrypsin or a collagenase.The biodegradable biopolymer material is preferably a porous substance.

The living cell-growth substrate according to the present inventionconsists of a biodegradable biopolymer material, which is silk fibroinfrom domesticated silkworm; silk fibroin from wild silkworm; a compositematerial comprising silk fibroin from domesticated silkworm and silkfibroin from wild silkworm; or a composite material comprising eithersilk fibroin from domesticated silkworm or silk fibroin from wildsilkworm and at least one secondary substance selected from the groupconsisting of cellulose, chitin, chitosan, chitosan derivatives, keratinfrom wool and polyvinyl alcohol. The substrate is used for effectivelyand economically growing living cells.

The biodegradable water absorbable material according to the presentinvention consists of a biodegradable biopolymer material, which is silkfibroin from domesticated silkworm; silk fibroin from wild silkworm; acomposite material comprising silk fibroin from domesticated silkwormand silk fibroin from wild silkworm; or a composite material comprisingeither silk fibroin from domesticated silkworm or silk fibroin from wildsilkworm and at least one secondary substance selected from the groupconsisting of cellulose, chitin, chitosan, chitosan derivatives, keratinfrom wool and polyvinyl alcohol.

The term “biodegradation” as used herein means any reaction including,for instance, a digestion or hydrolysis reaction of silk fibroin, and/orthe secondary substance into small molecules through the action of anenzyme and a digestion reaction into amino acids. Accordingly, an enzymemay degrade the substrate into small molecules through reactions otherthan digestion in the present invention, but the enzyme may convenientlybe referred to as a protease (proteolytic enzyme).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present invention, raw materials for use in the preparation of anaqueous solution containing silk fibroin from silk protein fibers may,for instance, be silk fibers from domesticated or wild silkworms. Thesilk fibroin of the silk fiber obtained from domesticated silkwormusable herein may be silk fibroin as a silk protein. For instance, thesilk protein may be from larvae of domesticated silkworm (Bombyx mori)reared in farmhouses and larvae of KUWAGO (Bombyx mandarina or mulberrywild silkworm), as a relative species of the domesticated silkworm.Examples of silk fibroin from wild silkworm usable herein are silkfibroin obtained from larvae of Antheraea pernyi, Antheraea yamamai,Antheraea militta, Antheraea assama, Philosamia cynthia ricini andPhilosamia cynthia pryeri. Alternatively, raw materials for preparingthe silk fibroin aqueous solution may be by-products from domesticatedand wild silkworms, silk fibers, silk fiber products and aggregates ofsilk fibers, in addition to the foregoing silk fibers.

As described above, the secondary substance to be hybridized with thesilk fibroin from domesticated or wild silkworm is at least one memberselected from the group consisting of cellulose, chitin, chitosan,chitosan derivatives, keratin from wool and polyvinyl alcohol.

An aqueous solution of silk fibroin from domesticated silkworm isadmixed with an aqueous solution of silk fibroin from wild silkworm.Alternatively, an aqueous solution of silk fibroin from domesticatedsilkworm or an aqueous solution of silk fibroin from wild silkworm isadmixed with an aqueous solution of the secondary substance. This isfollowed by extending the resulting mixed aqueous solution over thesurface of a substrate of, for instance, polyethylene, and thensolidifying the extended solution by drying it to produce abiodegradable biopolymer material. The biodegradable biopolymer materialproduced is a composite material (or a hybrid material) of the silkfibroin from domesticated silkworm and the silk fibroin from wildsilkworm, or a composite material of the silk fibroin from domesticatedsilkworm or the silk fibroin from wild silkworm with the secondarysubstance. Biodegradable biopolymer materials may be prepared from anaqueous solution containing only silk fibroin from domesticated silkwormand an aqueous solution containing only silk fibroin from wild silkwormby repeating the procedures used above.

The preparation of an aqueous solution of silk fibroin from domesticatedsilkworm, an aqueous solution of silk fibroin from wild silkworm, amembrane of silk fibroin from domesticated silkworm, and a membrane ofsilk fibroin from wild silkworm will be described in detail. A methodfor the preparation of a hybrid (composite material) using an aqueousmixed solution comprising an aqueous solution of silk fibroin fromdomesticated silkworm or an aqueous solution of silk fibroin from wildsilkworm and an aqueous solution of each secondary substance orcellulose, chitin, chitosan, chitosan derivatives, keratin from wool orpolyvinyl alcohol will also be detailed below.

(A) Preparation of Aqueous Solution of Silk Fibroin from DomesticatedSilkworm and Membrane of Silk Fibroin from Domesticated Silkworm

An aqueous solution of pure silk fibroin from domesticated silkworm maybe prepared by the following method:

First, cocoon fibers produced and spun by domesticated silkworm areboiled in an alkaline aqueous solution of a neutral salt, such as sodiumcarbonate to remove sericin, to prepare silk fibroin fibers as theentity of the domesticated silkworm silk fibers. Then, the resultingsilk fibroin fiber is dissolved in a concentrated aqueous solution of aneutral salt and heated to form a silk fibroin aqueous solution. Thissilk fibroin aqueous solution contains the silk fibroin and a largequantity of ions from the neutral salt. The aqueous solution is pouredinto a cellulose membrane for dialysis. Then, both ends of the membraneare tied up with sawing threads and dialyzed against tap water or purewater for a desired period of time ranging from 2 to 5 days to give anaqueous solution of pure domesticated silkworm silk fibroin. Aqueoussolutions of silk fibroin having a variety of concentrations can beprepared by partially evaporating the water of the resulting silkfibroin aqueous solution or by diluting the resulting silk fibroinaqueous solution with water.

The aqueous solution of domesticated silkworm silk fibroin thus preparedcan be extended over a substrate, such as a polyethylene membrane,followed by solidification of the extended layer of the silk fibroinsolution through evaporation to dryness at room temperature to give adomesticated silkworm silk fibroin membrane.

In addition, the domesticated silkworm silk fibroin aqueous solution canbe prepared by adding domesticated silkworm silk protein fibers (silkfibers) to a concentrated aqueous solution of a neutral salt, such ascalcium chloride, calcium nitrate, lithium bromide or lithiumthiocyanate, and then heating the mixture to dissolve the silk fibers inthe aqueous solution. The concentration of the neutral salt in theaqueous solution ranges from about 5 to 9M, and it is sufficient to heatthe mixture to a temperature ranging from about 25 to 70° C., preferably25 to 60° C., for the dissolution of the silk fibers. If the dissolutiontemperature exceeds 70° C., the molecular weight of the silk protein isreduced, the resulting material loses its polymeric characteristics,and, as a result, the molding properties of the material may beconsiderably impaired. The dissolution time is preferably 1 to 20minutes. Among the foregoing neutral salts, lithium salts satisfactorilydissolve the domesticated silkworm silk protein fibers and are excellentin solubilizing the domesticated silkworm silk fibroin fibers. Lithiumbromide is preferred. For instance, an aqueous lithium bromide solutionhaving a concentration of not less than 8M, and preferably not less than8.5M, would permit dissolution of domesticated silkworm silk proteinfibers by the treatment at a temperature of not less than 55° C. for atime of not less than 15 minutes.

(B) Preparation of Aqueous Solution of Silk Fibroin from Wild Silkwormand Membrane of Silk Fibroin from Wild Silkworm

To prepare an aqueous solution of wild silkworm silk fibroin from silkfibers from wild silkworms, such as those from Antheraea pernyi andAntheraea yamamai, the wild silkworm cocoon fibers are immersed in anaqueous solution of sodium peroxide in a predetermined amount withrespect to the mass thereof, and boiled for a desired time period toform wild silkworm silk fibroin fibers. The resulting silk fibroinfibers are dissolved in an aqueous solution of a neutral salt having ahigh solubilization ability. Then, the resulting aqueous solution isdialyzed in the same manner as the domesticated silkworm silk fiberabove to give an aqueous solution of pure wild silkworm silk fibroin.This preparation method is described in more detail below.

In the preparation of an aqueous solution of wild silkworm silk fibroinby dissolving wild silkworm silk fibers, the silk sericin covering thesurface of the wild silkworm cocoon fibers should be removed by a methoddifferent from the method used for refining the domesticated silkwormsilk sericin. This is because tannin is also adhered to the surface ofthe wild silkworm silk fibers and the sericin is insolibilized due tothe cross-linking action of the tannin. It is, for instance, necessaryto immerse the wild silkworm cocoon fibers in about 50 volumes of a 0.1%sodium peroxide aqueous solution on the basis of the mass of the cocoonfibers and to then subject the cocoon fibers to a boiling treatment, forinstance, at 98° C. for one hour, in order to remove the sericin andtannin. The wild silkworm silk fibroin fibers from which the sericin andtannin have been removed are then dissolved in an aqueous solution of aneutral salt having a high solubilization ability, such as lithiumthiocyanate. The solution of the wild silkworm silk fibroin fibers inthe aqueous neutral salt solution is poured into a cellulose membranefor dialysis. Then, both ends of the membrane are tied up with sawingthreads and dialyzed against tap water or pure water maintained at roomtemperature for a desired period of time ranging from 2 to 5 days tocompletely remove the lithium ions and to give an aqueous solution ofpure wild silkworm silk fibroin.

The aqueous solution of pure wild silkworm silk fibroin can be extendedover a substrate, such as a polyethylene membrane, followed bysolidification of the extended layer of the silk fibroin solutionthrough evaporation to dryness at room temperature to give a wildsilkworm silk fibroin membrane.

If the silk proteins from domesticated silkworm and those from wildsilkworm can be sufficiently admixed together in the form of aqueoussolutions, a composite material of these silk proteins may be preparedby solidifying an aqueous mixed solution of these components throughevaporation to dryness. The resulting hybrid membrane may possesscharacteristic properties, such as biodegradability, transparency,adhesion stability and other biochemical properties, different fromthose observed for the materials from domesticated silkworm silk fibroinalone and wild silkworm silk fibroin alone. We will explain the methodfor preparing a hybrid membrane of the domesticated silkworm silkfibroin and the wild silkworm silk fibroin starting from the aqueoussolutions of domesticated silkworm silk fibroin and wild silkworm silkfibroin.

(C) Hybrid Membrane of Domesticated Silkworm Silk Fibroin and WildSilkworm Silk Fibroin

Desired amounts of the aqueous solution of domesticated silkworm silkfibroin and the aqueous solution of wild silkworm silk fibroin preparedabove are digested into a beaker and carefully and gently mixed bystirring using a glass rod in such a manner that the aqueous solutionnever undergoes gelation. The mixed aqueous solution prepared can beextended over a substrate such as a polyethylene membrane, followed bysolidification of the extended layer of the silk fibroin solutionthrough evaporation to dryness at room temperature to give a transparenthybrid membrane. The concentrations of the aqueous solution ofdomesticated silkworm silk fibroin and the aqueous solution of wildsilkworm silk fibroin are preferably on the order of 0.1 to 3% w/v andparticularly preferably 0.4 to 2% w/v, respectively.

The blending of an aqueous solution of domesticated silkworm silkfibroin or an aqueous solution of wild silkworm silk fibroin with anaqueous solution of a secondary substance as detailed below may becarried out in the same manner used above.

In this respect, a method for the preparation of a hybrid ofdomesticated silkworm silk fibroin and silk fibroin from Antheraeapernyi has already been reported by the inventors of this invention (M.Tsukada et al., Journal of Applied Polymer Science, 1994, 32:1175-1181). However, this article does not include any description,which teaches and/or suggests the biodegradability of the hybrid.

(D) Hybrid Membrane of Domesticated or Wild Silkworm Silk Fibroin andCellulose

A hybrid membrane consisting of domesticated silkworm silk fibroin andcellulose can be prepared by admixing the foregoing aqueous solution ofthe domesticated silkworm silk fibroin and an aqueous solution ofcellulose according to the following method.

First, domesticated silkworm silk fibroin fibers and commerciallyavailable powdery cellulose (available from Fluka Company) free of anyparticular purification treatment are separately dissolved incuprammonium ([Cu(NH₃)₄](OH)₂) aqueous solution to form respectiveaqueous solutions. Then, these two aqueous solutions are admixed in adesired mixing ratio (domesticated silkworm silk fibroinfibers/cellulose) by careful and gentle stirring in such a manner thatthe mixture never undergoes any gelation, precipitation and/orsolidification. The mixed aqueous solution thus prepared is gentlyextended over a substrate such as a glass plate placed on a horizontalplane. A mixed solution containing acetone and acetic acid is thencarefully added to the surface of the extended mixed aqueous solution toremove the metal complex present in the mixed aqueous solution whilesolidifying the domesticated silkworm silk fibroin and cellulose.Thereafter, the solidified mixture is washed with a mixed solution ofglycerin and water and then with water, followed by drying the mixtureat room temperature to give a hybrid membrane containing domesticatedsilkworm silk fibroin and cellulose.

A hybrid membrane containing wild silkworm silk fibroin can be preparedby the same procedures used above in the preparation of the hybridmembrane containing the domesticated silkworm silk fibroin.

In this respect, the inventors of this invention and the collaboratorshave already reported a method for the preparation of a hybrid ofdomesticated silkworm silk fibroin and cellulose. (see G. Freddi et al.,Journal of Applied Polymer Science, 1995, 56: 1537-1545). However, thisarticle does not include any description, which teaches and/or suggeststhe biodegradability of the hybrid.

(E) Hybrid Membranes of Domesticated or Wild Silkworm Silk Fibroin andChitin, Chitosan and Chitosan Derivatives

A hybrid membrane comprising domesticated silkworm silk fibroin andchitin, chitosan or a chitosan derivative can be prepared by admixing anaqueous domesticated silkworm silk fibroin solution and an aqueoussolution of chitin, chitosan or a chitosan derivative according to thefollowing method. The chitosan derivative usable in the presentinvention is not restricted to any specific one and may be, forinstance, chitin, carboxylated carboxy methyl chitin (also referred toas “CMK”) from chitosan, Na salt of carboxy methyl chitin and glycolchitosan.

The chitin used in the present invention may be, for instance, from amarine crustacean, such as a prawn or a black tiger, or chitin coveringthe crust of an insect. The crust of a crustacean or an insect comprisesinorganic substances, such as calcium carbonate and proteins. Therefore,chitin may be isolated by the removal of contaminants other than chitinaccording to any currently known method. Moreover, it is also convenientto use a commercially available powdery product of chitin (Wako PureChemical Industries, Ltd.).

Chitin may be converted into water-soluble one according to thefollowing method. First, powdery chitin is suspended in a concentratedaqueous caustic alkali (such as sodium hydroxide) solution and stirredover a desired period of time under reduced pressure. Then, theresulting powdery chitin is charged into a concentrated aqueous causticalkali solution containing a surfactant such as sodium dodecyl sulfate,stirred and allowed to stand overnight at a low temperature (forinstance, −20° C.). Alternatively, the resulting powdery chitin issuspended in liquid ammonia (−33° C.) and then metal potassium is addedto the resulting suspension to prepare alkali chitin in which thehydrogen atoms on the C6 and C3 hydroxyl groups of chitin aresubstituted with sodium or potassium. The alkali chitin prepared iscompressed and dispersed in ice crushed into fine pieces, followed bythe sulfidation of the chitin through the addition of carbon disulfideto obtain a chitin sulfide. An aqueous solution can be prepared usingthis sulfide.

Moreover, the alkali chitin may be reacted with an epoxy compound, anallyl or an alkali halide to give an o-allyl derivative or an o-alkylderivative. Further, the alkali chitin may be reacted with ethylenechlorohydrin (2-chloroethanol) to give ethylene glycol chitin or withchloroacetic acid to give o-(carboxymethyl) chitin. If ethylene glycolchitin is reacted with a concentrated caustic alkali aqueous solution(for instance, a 40% sodium hydroxide aqueous solution) under desiredreaction conditions (for instance, 100/C for 5 hours) with stirring, theacetamide groups present on the chitin molecules are hydrolyzed intofree amino groups to give water-soluble glycol chitosan. Chitosanderivatives including the glycol chitosan can be easily dissolved in anaqueous acid solution having a wide concentration range, such as anaqueous acetic acid solution.

A mixed aqueous solution obtained by the addition of a domesticatedsilkworm silk fibroin aqueous solution to the foregoing aqueous glycolchitosan solution may be extended over, for instance, a polyethylenesubstrate, and then solidified through evaporation to dryness to preparea transparent and soft composite material (a hybrid membrane) comprisingdomesticated silkworm silk fibroin and glycol chitosan. Hybrid membranesmay likewise be prepared by the use of aqueous solutions of otherchitosan derivatives or an aqueous solution of water-solubilized chitin,instead of the foregoing glycol chitosan aqueous solution, according tothe same procedures used above.

In the case of wild silkworm silk fibroin, hybrid membranes may beprepared by repeating the procedures used above in connection with thedomesticated silkworm silk fibroin.

(F) Hybrid Membrane of Domesticated or Wild Silkworm Silk Fibroin andWool Keratin

Hybrid membrane of domesticated or wild silkworm silk fibroin and woolkeratin usable in the present invention may be, for instance, woolkeratin fibers as well as aqueous keratin solutions and aqueousS-carboxy methyl keratin (CMK) solutions, which can be prepared asfollows. These aqueous solutions may be prepared according to theconventionally known methods.

To solubilize wool yarns, the Cystine cross linkings are cleaved using areducing agent (such as mercapto-ethanol or thioglycollic acid) in anitrogen gas atmosphere or keratin molecules are reduced andsolubilized. If mercapto-ethanol is used, it is preferred to carry outthe reduction in a urea solution. In this case, the concentration ofurea ranges from 7.5 to 8.8 M, preferably 7.8 to 8 M. If thioglycollicacid is used, it is desirable to add NaCl to the reaction system in anamount of 1 to 4%.

When using mercapto-ethanol, which may act as a reducing agent, woolyarns are immersed in a urea solution having a concentration specifiedabove, followed by degassing. Mercapto-ethanol is added to the mixturein an amount of 3 to 5 mL per 10 g of wool yarns at a temperature of notmore than 45° C., desirably 20 to 25° C., in a nitrogen gas atmosphere.The resulting mixture is stirred over a predetermined period of time(for instance, about 3 hours). Thus, keratin molecules in wool yarns arereduced and keratin molecules having SH groups are prepared. Then, thereaction system containing the keratin molecules having SH groups isdigested into a cellulose membrane for dialysis. Next, both ends of thecellulose membrane are tied up with sawing threads and sufficientlydialyzed against pure water to remove the urea and the excessmercapto-ethanol to give an aqueous solution of the wool keratin. Thisaqueous wool keratin solution may be used as an aqueous solution of asecondary substance used in the present invention according to the sameprocedures used above.

Moreover, if the wool keratin carrying —SH groups obtained above isfurther reacted with an alkylation agent, for instance, any knownalkylation agent such as an (substituted) alkyl halide to form anS-(substituted) alkyl keratin, the aqueous solution thereof may likewisebe used in the present invention. This alkylation may be carried outaccording to any known method. The alkylation will be described belowusing iodoacetic acid as an alkylation agent by way of example. To theforegoing reduced keratin, iodoacetic acid is added (molecular weight:185.95) in an amount ranging from 10 to 17 g per 10 g of the wool yarnsin order to react them at a temperature ranging from 20 to 25° C. in anitrogen gas atmosphere with stirring. After 1 to 2 hours, the pH valueof the reaction system is adjusted to about 8.5, followed by dialysisagainst pure water to remove the excess iodoacetic acid to give anaqueous solution of S-carboxymethyl keratin.

To the aqueous solution of the reduced keratin or the aqueous solutionof the S-carboxymethyl keratin, there can be added an aqueous solutionof domesticated silkworm silk fibroin to give a mixed aqueous solution.The mixed aqueous solution is extended over the surface of a substratesuch as a polyethylene substrate, and then the extended aqueous layer isdried to give a hybrid membrane of the reduced keratin, or theS-carboxymethyl keratin and the domesticated silkworm silk fibroin.

In the case of the wild silkworm silk fibroin, a hybrid membrane can beprepared according to the same procedures used for preparing the hybridmembrane of the domesticated silkworm silk fibroin.

(G) Hybrid Membrane of Domesticated Silkworm Silk Fibroin or WildSilkworm Silk Fibroin and Polyvinyl Alcohol

Polyvinyl alcohol (PVA having an average degree of polymerization ofabout 2000 available from Wako Pure Chemical Co., Ltd.) is charged intohot water, followed by careful dissolution using a stirring machine toform an aqueous PVA solution having a desired concentration (forinstance, a 0.5% w/v PVA aqueous solution). An appropriate amount of anaqueous solution of domesticated silkworm silk fibroin is added to thisPVA aqueous solution. The resulting mixture is allowed to stand at roomtemperature for not less than 30 minutes to form a complex aqueoussolution of domesticated silkworm silk fibroin and PVA. The complexaqueous solution can be extended over the surface of a substrate, suchas a polyethylene substrate, and the moisture of the extended aqueouslayer is evaporated over a whole day and night to give a transparenthybrid membrane of PVA and the domesticated silkworm silk fibroin.

In the case of the wild silkworm silk fibroin, a hybrid membrane can beprepared according to the same procedures used for preparing the hybridmembrane of the domesticated silkworm silk fibroin.

The inventor of this invention and the collaborators have alreadyreported a method for the preparation of a hybrid membrane of PVA anddomesticated silkworm silk fibroin (see, M. Tsukada et al., Journal ofApplied Polymer Science, 1994, 32: 243-248). However, this article doesnot include any disclosure that refers to or suggests thebiodegradability of the hybrid membrane.

As discussed above, the silk proteins from domesticated silkworm, wildsilkworm and secondary substances may be admixed together in theiraqueous solution states and hybrid membranes can be prepared from theresulting aqueous mixed solutions. The resulting hybrid membranes mayshow biochemical characteristic properties such as biodegradability,transparency (light transmission properties) and a cell-growth ability,which are different from those observed for a material simply comprisingdomesticated silkworm silk fibroin or wild silkworm silk fibroin. Inaddition, the hybrid membrane also possesses, for instance, excellentmetal ion-adsorbing properties and resistance to peeling. To obtain ahybrid membrane from an aqueous solution of domesticated silkworm silkfibroin, an aqueous solution of wild silkworm silk fibroin, and aqueoussolutions of secondary substances in this case, it is sufficient thatthe concentration of each aqueous solution falls within the range offrom 0.1 to 5% w/v, as specified above, preferably 0.4 to 3% w/v, toobtain hybrid membranes having uniform quality. In this respect, theaqueous solution of domesticated silkworm silk fibroin and the aqueoussolution of wild silkworm silk fibroin; and the aqueous solution ofdomesticated silkworm silk fibroin or the aqueous solution of wildsilkworm silk fibroin and the aqueous solution of secondary substancesmay be admixed together in any rate. Therefore, the mixing ratio ofthese components in the resulting composite may, if desired, be set atan arbitrarily level.

To admix domesticated silkworm silk fibroin and wild silkworm silkfibroin, or an aqueous solution of domesticated silkworm silk fibroin oran aqueous solution of wild silkworm silk fibroin with an aqueoussolution of a secondary substance, it is sufficient to gently admixthese aqueous solutions by stirring with a glass rod. If these solutionsare admixed rapidly, vigorously or violently, a shear stress is appliedto the silk fibroin molecules, the aqueous solutions undergo coagulationand it is sometimes observed that these solutions are not uniformlyadmixed.

The biodegradable biopolymer material of the present invention may haveany shape, such as a sheet-like, membrane-like, powdery, bead-like,gel-like, fibrous, tubular or hollow thread-like shape.

In the present invention, the biodegradability of a biodegradablebiopolymer material can be evaluated by treating it with a bufferingsolution containing a peptidase in a predetermined concentration for apredetermined period of time. More specifically, the biodegradablebiopolymer material is digested (or hydrolyzed) through the treatmentthereof with an enzyme-containing aqueous dissociation solution preparedby dissolving an enzyme having a desired activity in a desired bufferingsolution at 37° C. for a predetermined period of time. The degree ofbiodegradation is evaluated by calculating the extent of thebiodegradable biopolymer material digested by the enzyme on the basis ofthe weight change of the sample.

The degree of digestion is greatly influenced by the kinds of enzymesused, the concentrations of the enzyme, the time required for theenzyme-decomposition and/or the kinds of materials to be treated.Moreover, the degree of digestion also greatly varies depending onwhether the material is silk protein fibers or silk protein membranes.The silk protein fiber produced by silkworm has a fibrous structurepeculiar thereto and a large density of hydrogen bonds formed betweenfibrous molecules. Therefore, it is hardly hydrolyzed, even whenintroducing it into an aqueous solution of a peptidase. For this reason,the silk protein fiber can be used as a sample for a biodegradation testwithout any pre-treatment. In contrast, a silk fibroin membrane, or thelike as a silk protein membrane prepared after once dissolving the silkprotein fibers in a neutral salt solution, will swell through theabsorption of moisture and will ultimately dissolve. In thebiodegradation test, the dissociation behavior of the material in abuffering solution containing an enzyme is examined. Therefore, the silkfibroin membrane prepared cannot be directly subjected to such abiodegradation test. It is necessary to subject the membrane to aninsolubilization treatment in order to use it as a test sample. Thematerial or membrane may be insolibilized by, for instance, immersion inan aqueous solution of an alcohol, such as methanol or ethanol; or bythe use of a conventionally known epoxy compound or an aldehyde, such asformalin. For instance, the membrane may be insolibilized by immersingit in a 20 to 80% methanol aqueous solution for a time usually rangingfrom 5 to 10 minutes, preferably a 40 to 60% methanol aqueous solutionfor 5 to 10 minutes. More specifically, it is sufficient to lightlyimmerse the membrane in a 50% (v/v) methanol aqueous solution at roomtemperature for not less than one minute and then dry it in air at roomtemperature.

Moreover, almost all of the composite materials, other than theforegoing silk fibroin membrane, are insoluble in water immediatelyafter preparation by the process for evaporation to dryness. Usually,these materials are desirably insoluble in water in many applications.It is sufficient, in such cases, to make them insoluble in water by thetreatment with methanol. The composite material of domesticated silkwormsilk fibroin and cellulose or that of domesticated silkworm silk fibroinand polyvinyl alcohol is water-soluble immediately after the preparationthereof. If the composite material is treated with methanol, the silkfibroin becomes insoluble in water. However, the cellulose and polyvinylalcohol components are not converted into water-insoluble componentsthrough such a methanol treatment. Accordingly, it is preferred tosubject such composite materials to a cross-linking reaction with areagent having a strong cross-linking ability such as formalin.

Any peptidase (digestive enzyme) may be used in the present invention.The peptidase may be one which cleaves a distinct site of a substrate orone whose cleaving site on a substrate cannot be specified. Thebiodegradable biopolymer material of the present invention may bebiodegraded by the action of an enzyme such as proteases, collagenases,and chymotrypsin. As described above, it is desirable for the evaluationof the biodegradability using these enzymes to use a buffering solutionhaving a desired pH value capable of maintaining the maximum enzymeactivity. No specific combination of an enzyme and a buffering solutionused in the enzymatic decomposition is required. Examples of preferredcombinations of enzymes and buffering solutions are a collagenase and 50mM TES (buffering solution) or 50 mM CaCl₂ (pH 7.4); chymotrypsin and 50mM Tris (buffering solution) or 5 mM CaCl₂ (pH 7.8); and a protease and40 mM potassium phosphate (buffering solution) (pH 7.5). A boratebuffering solution having a low ionic strength is preferably used assuch a buffering solution and the pH thereof roughly ranges from 7 to 8.

The concentration of the protein hydrolase (or peptidase) aqueoussolution may vary depending on the kinds of proteins used as substratesand in general ranges from 0.1 to 0.8 mg/mL, preferably 0.2 to 0.5mg/mL. If the enzyme concentration is less than 0.1 mg/mL, theefficiency of the digestion is insufficient. If it exceeds 0.8 mg/mL,the biodegradation experiment becomes less advantageous from aneconomical standpoint.

One of the inventors of this invention has previously prepareddomesticated silkworm silk fibroin membrane and domesticated silkwormsilk fibers by dissolving and biodegrading domesticated silkworm silkfibroin fibers, to clarify the biodegradation behavior thereof over time(see N. Minoura et al., Biomaterials, 1990, 11 (August): 430-434). Inthis article, it is confirmed that domesticated silkworm silk fibroinmembrane is hydrolyzed to a significant extent in a protease solution,while the domesticated silkworm silk fibers are not hydrolyzed to anysignificant degree. However, silk material from wild silkworm contains asilk protein having a primary structure completely different from thechemical structure of the domesticated silkworm silk fibers. There hasnot yet been reported any information concerning the biodegradability ofwild silkworm silk fibers and wild silkworm silk fibroin membrane.

According to the present invention, a powdery biodegradable biopolymermaterial can be prepared by lyophilizing an aqueous solution ofdomesticated silkworm silk fibroin, an aqueous solution of wild silkwormsilk fibroin, an aqueous mixed solution containing an aqueous solutionof domesticated silkworm silk fibroin and an aqueous solution of wildsilkworm silk fibroin or an aqueous mixed solution comprising either anaqueous solution of domesticated silkworm silk fibroin or an aqueoussolution of wild silkworm silk fibroin and an aqueous solution of asecondary substance, such as cellulose, according to any known method.More specifically, these aqueous solutions are frozen at a temperatureof about −10° C. Then, the frozen solutions are allowed to stand in anatmosphere maintained at a reduced pressure to remove the moisturepresent in the sample and to thus form a powdery material. In addition,a gel-like biodegradable biopolymer material may be obtained byadjusting the pH value of the aqueous solution of each sample to fallwithin the acidic region, for instance, not more than 4.4, to coagulatethe entire aqueous solution and convert it into a gel. A membrane-likebiodegradable biopolymer material may be obtained by extending theaqueous solution of each sample over a substrate, such as a polyethylenesubstrate or a glass plate, followed by evaporating the extended layerto dryness for a sufficient period of time.

All of the foregoing powdery, gel-like and membrane-like biodegradablebiopolymer materials are soluble in water. Therefore, they can, ifdesired, be insolibilized in water by immersing in an aqueous alcoholsolution as discussed above.

The biodegradability of the biodegradable biopolymer material of theinvention through the action of a hydrolase is determined by theconcentration of the enzyme, the buffering solution used, the digestiontime, the degree of water-insolubilization and the content of thedomesticated silkworm silk fibroin. For this reason, thebiodegradability of a material can be improved by reducing thewater-insolubility or increasing the water-solubility, and increasingthe content of the domesticated silkworm silk fibroin in the material. Asilk material free of any fibrous structure, such as silk fibroinmembrane, is quite susceptible to digestion with an enzyme, in contrastto the silk fibroin fibers. In particular, the biodegradability of acomposite material (hybrid) is determined by the degree of waterinsolubility of the domesticated and wild silkworm silk fibroins, thesecondary substance selected, the mixing ratio of the domesticated orwild silkworm silk fibroin to the secondary substance, the enzymeselected, the enzyme concentration and the treating time. Therefore, theconditions for preparing hybrids, the mixing ratios or thebiodegradation conditions can be appropriately changed or selecteddepending on the desired purposes.

A biodegradable biopolymer material having a good biocompatibility canbe prepared by hybridizing or blending silk fibroin with an organicpolymer (secondary substance). An organic polymer is excellent in theaffinity to biological tissues, but is hardly decomposed with a proteinhydrolase.

The biodegradable biopolymer material of the present invention may be ahybrid of materials, both of which serve as substrates for enzymes, suchas proteases, collagenases and chymotrypsin; or a hybrid of a polymermaterial capable of serving as a substrate and a secondary substance,which cannot serve as a substrate. Examples of proteins capable ofserving as substrates for these three kinds of enzymes are domesticatedsilkworm silk fibroin, wild silkworm silk fibroin and wool keratin. Whenhybridizing these materials capable of serving as substrates for theenzymes with naturally occurring polymers, which cannot serve assubstrates of these enzymes, such as cellulose, chitin, chitosan,chitosan derivatives and polyvinyl alcohol, there is observed a tendencythat the amount of the hybrid biodegraded is gradually reduced as thecontent of the naturally occurring polymer in the hybrid increases.

For instance, in the case of a hybrid membrane consisting ofdomesticated silkworm silk fibroin and cellulose, the domesticatedsilkworm silk fibroin is easily decomposed by the action of a protease.Therefore, the higher the content of the domesticated silkworm silkfibroin, the easier the control of the degree of biodegradation of thehybrid. However, the behavior of the domesticated silkworm silk fibroinfor a cellulase is completely contrary to the behavior discussed above.Accordingly, the higher the content of the cellulose, the smaller theamount of the hybrid biodegraded as a whole. Thus, a biodegradablebiopolymer material having a desired rate of biodegradation can beprepared by changing the mixing ratio of the protein capable of servingas a substrate for an enzyme used to a secondary substance, which cannever serve as a substrate for the enzyme.

The biopolymer usable herein is not restricted to any specific one andmay be, for instance: silk proteins from domesticated and wild silkworms(such as silk fibroins and silk sericin) or keratins from animals (suchas wool keratin); collagen; and gelatin. The present invention may use,for instance, silk proteins from domesticated and mulberry wildsilkworms, or silk proteins from Antheraea yamamai, Antheraea pernyi,Philosamia cynthia ricini and Philosamia cynthia pryeri Silkworms aswild silkworms. Such biopolymers may be silk fibers, silk fiber productsfrom domesticated and wild silkworms or fibrous aggregates thereof, orkeratin fibers from animals and keratin fiber products.

The biodegradable biopolymer material of the present invention is usefulas a metal ion-adsorbing material. In particular, when immersing acomposite material (hybrid) as a biodegradable biopolymer material ofthe present invention in an aqueous solution containing antibacterialmetal ions, such as silver, copper and/or cobalt ions, the compositematerial adsorbs a large quantity of these metal ions. Therefore, thecomposite material carrying metal ions can be useful as an antibacterialmaterial. Alternatively, when immersing the biodegradable biopolymermaterial in waste water, the material adsorbs various kinds of metalions present in the waste water (for instance, base metal ions, such asCu²⁺, Ni²⁺, Vo²⁺, Zn²⁺, Co²⁺ and Al³⁺, and ions of rare earth metals,such as Yb, Nd, Pr and La). Accordingly, the material is also useful asa material for adsorbing metal ions present in waste water. The metalions adsorbed on the material may be recovered or disposed, according tocircumstances.

A useful substance, such as a water-soluble medicine or apharmaceutically active substance, can be included in or immobilized onthe biodegradable biopolymer material, in particular, the compositematerial of the present invention. The resulting product may beimplanted or embedded in, for instance, a living body, so that theproduct implanted may gradually release the medicine or pharmaceuticalcomponent, while the material is decomposed and/or deteriorated throughdigestion with, for instance, a protease present in the body fluid.Therefore, the material of the present invention can be used as asustained release carrier for useful substances. In this respect, thesilk fibroin fiber from domesticated or wild silkworm may be used tomake the biodegradability thereof light, or to make a membrane-likesample, which is obtained by dissolving domesticated or wild silkwormsilk fibers using a neutral salt, desalting the resulting solution usinga cellulose dialysis membrane and then evaporating the dialyzed solutionto dryness to obtain an easily decomposable material. The membrane ofdomesticated silkworm silk fibroin is more easily biodegraded than themembrane of wild silkworm silk fibroin. Therefore, it is sufficient toincrease the content of the wild silkworm silk fibroin to form a hardlybiodegradable composite material comprising domesticated and wildsilkworm silk fibroins.

As described above, when using the biodegradable biopolymer material, inparticular, the composite material of the present invention whileembedding it in the living body, the material is ultimately decomposedinto small molecules, such as water and carbon dioxide, by the action ofenzymes present in the body, such as proteases, chymotrypsin andcollagenases, and finally excreted outside the body. A hybrid membranewith easily biodegradable domesticated silkworm silk fibroin may bebiodegraded within a relatively short period of time even when embeddingthe same in the living body unlike hardly biodegradable domesticatedsilkworm silk fibroin fibers. Therefore, the hybrid may be used for thetemporal assist of the healing of remediable damaged biological tissuesor as a sustained release carrier for drugs as discussed above. Such invivo degradable and absorbable material may be used in a variety ofapplications, such as the suture of incised and/or wound portions,arrest of hemorrhage, bone fixation, a clue for tissue-regeneration anda means for preventing adhesion.

The hybridization of domesticated or wild silkworm silk fibroin with asecondary substance would provide such a conspicuous effect that theresulting hybrid shows, on it surface, excellent biochemical properties,which have never been observed for the surface of the domesticated orwild silkworm silk fibroin or the secondary substance. For instance, therate of cell-growth on the surface of the hybrid is higher than thatobserved on the surface of a product consisting simply of domesticatedor wild silkworm silk fibroin or a secondary substance. Moreover, thehybridization of domesticated silkworm silk fibroin with wild silkwormsilk fibroin or the hybridization of a secondary substance, such ascellulose, with domesticated or wild silkworm silk fibroin would providea hybrid or composite material having improved moldability andtransparency, as compared with those observed for a membrane simplyconsisting of domesticated or wild silkworm silk fibroin, and wouldpossess excellent cell adhesion properties. In addition, the compositematerial has a high wear resistance and the rate of cell-growth on thecomposite surface is improved, as compared with that observed on thesurface of a membrane consisting of a single protein. Accordingly, sucha composite material may be used as cell-growth materials in the fieldof biochemistry.

Moreover, cellulose derivatives may be used in food additives,cosmetics, additives for drugs and pharmaceutical preparations, such asanti-thrombotic agents. Therefore, the composite materials consisting ofdomesticated silkworm silk fibroin and cellulose may be used inapplications similar to those for the cellulose.

The biodegradable biopolymer material of the present invention possesseswater-absorbing properties, which make the material applicable as awater-absorbable resin used in, for instance, disposable hygienic goodsand household goods, water cut-off agents, soil conditioners, dewinginhibitors, and water-retention agents for agriculture and horticulture.The present invention would also permit the supply of a water-absorbingmaterial having such biodegradability for a low price without requiringany complicated steps. For this reason, the material of the presentinvention can be applied to any fields of applications identical tothose for the conventionally known water-absorbing resins. For instance,the material of the present invention can be used in a wide variety offields, such as hygiene (typically use as a diaper and a sanitary good),medical service (for instance, use in cataplasms), civil engineering andarchitecture (for instance, use as an agent for gelling sludge), foods,industries, and agriculture and horticulture (for instance, use as asoil conditioner and a water-retention agent).

The present invention will be described below in more detail withreference to the following Examples and Comparative Examples, but thepresent invention is not limited to these specific Examples. In thefollowing descriptions, the term “%” means w/v unless otherwisespecified.

Various test methods used in the following description will be describedin detail.

(1) Evaluation of Mechanical Properties

The strength and elongation at the break of each silk fiber upon thebreakage thereof were determined using INSTRON (Autograph AGS-5Davailable from Shimadzu Corporation) under the following measurementconditions: the length of a sample to be tested of 50 mm, the rate ofextension of 10 mm/min and the chart full scale of 250 g. In thisrespect, each measured value means the average of 20 measurementsrepeatedly carried out.

(2) Methods for the Adsorption of Metal Ions on Biodegradable BiopolymerMaterial and for Quantitative Determination thereof

Each sample to be examined was immersed in a 0.5 mM aqueous metal saltsolution containing potassium nitrate (the pH value thereof was adjustedto 11.4 by the addition of aqueous ammonia) at room temperature for 30hours to adsorb metal ions on the sample. In this respect, metal ionswere adsorbed on the sample by immersing the latter in an aqueous metalsalt solution (the pH value thereof was controlled to 8.5).

The metal ions adsorbed on each test sample were analyzed using a plasmaatomic absorption spectrometer (ICP-AES) available from Perkin-ElmerCompany. More specifically, each test sample (5 to 10 mg) was completelyhydrolyzed with 2 mL of a 65% aqueous nitric acid solution in amicrowave hydrolysis furnace (MDS-81DCCEM), 10 mL of water was added tothe hydrolyzed sample prior to the analysis and then the resultingmixture was subjected to the analysis in the ICP-AES. The amount ofmetal ions adsorbed on each sample is expressed in terms of the amountof metal ions (in mM unit) per unit mass of the sample.

(3) Decomposition Treatment with Enzyme

An enzyme used in the biodegradation experiment was dissolved in abuffering solution optimum for the digestion. This solution was chargedinto a 100 mL volume sterilized glass beaker, followed by the additionof each test sample and decomposition thereof with the enzyme at 37° C.for a predetermined time. The degree of biodegradation of each testsample observed after the treatment over a predetermined time isexpressed in terms of the rate of the residual sample (by weight)(hereunder referred to as “rate of remaining weight”) irrespective ofthe presence of the enzyme. More specifically, the rate of remainingmass is given by the following equation: [(Wi−We)/Wi]H 100(%) wherein Wiand We represent the masses of each sample determined before and afterthe biodegradation test, respectively.

Thus, the term “rate of remaining mass” herein used means the rate (%)of the residual sample (by weight), even after the digestion to thesample weight prior to the biodegradation. The smaller the value, thegreater the amount of the sample hydrolyzed or the higher thebiodegradability of the sample.

(4) Biodegradation Rate

The digestion rate observed when hydrolyzing each test sample with anenzyme was evaluated by the following method. The digestion rate isherein defined to be the amount (%) of the sample biodegraded during thebiodegradation procedure carried out over 50 hours relative to theinitial mass of each test sample or the mass of the test sample at theinitiation of the biodegradation test, which is defined to be 100.Therefore, the higher the digestion rate observed for a specific sample,the higher the biodegradability of the sample.

(5) Fourier Transform Infrared Absorption Spectra

The absorption spectra of each test sample concerning the molecularshape thereof were analyzed using an FT-IR (Fourier Transform InfraredAbsorption Spectra) measuring device available from Perkin-ElmerCompany. This analysis was carried out over a wave number range of from2000 to 400 cm⁻¹ and the number of scanning was 20.

(6) Test for Wear Resistance

A GAKUSHIN Type color fastness to rubbing tester Model II was used as arub tester. A polyethylene terephthalate (PET) substrate coated with athin membrane of each sample, selected from a variety of silk proteins,was fitted to the rub tester. The wear resistance test was carried outby reciprocating a friction element having an applied load of 500 g over10 times in such a manner that the sample fitted to the friction elementis lightly rubbed with that fixed to a test table under the action of apredetermined load. The sample was subjected to the FT-IR spectroscopicmeasurement before and after the wear resistance test to determine thewave numbers of absorption peaks. A sample thin membrane on the PETsubstrate whose FT-IR absorption peak shows reduction of its intensityafter the friction operation is judged to be easily peelable.

(7) Crystallinity Index of Domesticated Silkworm Silk Fibroin Membrane

The crystallinity index of a domesticated silkworm silk fibroin membranehydrolyzed with a variety of enzymes was evaluated according to thefollowing method: In this method, the Fourier transform infraredabsorption spectra (FT-IR) measuring device used was a Nicolet-150Pmeasuring device available from Nicolet Instruments, Madison, Wis.equipped with an ATR diamond cell (SPECAC). In the IR spectroscopy, thepeak strengths of amide band III at wave numbers of 1230 cm⁻¹ and 1260cm⁻¹ were determined and the crystallinity index (CI) was determinedaccording to the following formula. In this respect, the value of CI isa numerical value corresponding to the ratio of the peak strengths anddoes not have any unit.CI═I[1230 cm⁻¹]/I[1260 cm⁻¹](8) Amino Acid Analysis

Various protein materials corresponding to various biodegradation timeswere subjected to the following amino acid analysis. Each test sampleused herein was prepared by hydrolyzing each protein material with a 6Nhydrochloric acid solution at 105° C. for 24 hours. The amino acidanalysis was carried out using RP-HPLC.

(9) Test for Antibacterial Activity Against Vegetative PathogenicBacteria:

As vegetative pathogenic bacteria, the bacterial canker of tomato(scientific name: Corynebacterium michiganense pv. michiganense) wasselected. This bacterial canker is typical of the universal vegetativepathogenic bacteria, whose resistant bacteria may be easily induced andmay attack various kinds of plants, or which is a polyxeny putrefactivebacterium and is one of the rare gram positive bacteria in thevegetative pathogenic bacteria. The antibacterial activity of each testsample (composite membrane) was evaluated on the basis of thegrowth-inhibitory effect thereof on the vegetative pathogenic bacterium.

The evaluation of antibacterial effects in the following Examples wascarried out according to the following method.

Method for Examining Antibacterial Activity against Bacterium

25 mL of semi-synthesized Wakimoto Medium or King Medium B, which hadbeen dissolved with heating and then maintained at 55° C., was admixedwith 2 mL of the bacterium to be assayed (concentration: 109/mL). Themixture was poured into a petri dish to solidify the same in aplate-like shape. A sample membrane of about 1 cm square was placed onthis plate-like medium containing the bacterium and the whole sample wasclosely adhered to the culture medium. The resulting assembly wasmaintained at a temperature ranging from 20 to 25° C. The size of theinhibitory circle appearing at the periphery of the sample waspractically determined in the unit of mm in predetermined intervals toevaluate the bacterial growth-inhibitory effect observed on the culturemedium in the proximity to the sample to be assayed and to thus confirmthe presence of any antibacterial activity or evaluate the relativesuperiority on the basis of the change in the size of the inhibitorycircle observed.

(10) Test for Insect's Cell Growth

Ae cells from Antheraea pernyi or Bm cells from domesticated silkwormwere cultivated using a culture medium comprising Grace Medium (G8142available from Sigma Company) containing 5% powdered body fluid ofsilkworm and 5% fetal calf serum (available from Gibco Company), towhich 1% penicillin-streptomycin mixed antibiotic had been supplemented.After 2 days, the number of insect cells present in the cell culturemedium was determined using a hemocytometer to analyze the conditions ofthe Ae and Bm cells proliferated on the surfaces of various kinds ofcomposite materials whose silk fibroin contents were different from oneanother.

(11) Determination of Molecular Weight

Each fibrous or membrane-shaped sample used in the biodegradation testwas dissolved in a small amount of a 50% (w/v) aqueous solution oflithium thiocyanate at 40° C. over 30 minutes. Then, the resultingsolution was diluted with 50 mM sodium phosphate buffering solution, a0.15 M aqueous solution of potassium chloride (pH 7.2) and a 5M aqueoussolution of urea. The diluted solution was digested into a dialysismembrane of cellulose (Spectra/Por 6, MW 10=3.5 kDa, available fromSpectrum Company). Then, the solution in the membrane was dialyzedagainst the same buffering solution over 48 hours. After the dialysis,the dialyzate was diluted with distilled water to a silk fibroinconcentration of 1 mg/mL, filtered through a 0.2:m porous filterimmediately thereafter, and then analyzed by the size exclusionchromatography technique. Waters chromatographic system used herein isequipped with a pump (mod. 510) provided with a temperature controldevice, an injector (mod. U6K) and a refractive index detector (mod.410). This system is provided with software for chromatography (Maxima820 (Waters)) and GPC Lanter software. The column temperature was set at30/C. The column used herein was Shodex Protein KW-804 (Waters, 8 H 300mm) packed with porous silica gel coated with hydrophilic OH groups(pre-column, Shodex Protein KW-G, 6 H 50 mm). The amount of the sampleloaded on the column ranged from 50 to 100:1 and the exit velocity wasset at 0.5 mK/min. The analysis was carried out using distilled water asthe moving phase and optimally using a 50 mM sodium phosphate bufferingsolution, a 0.15 M aqueous solution of potassium chloride (pH 7.2) and a5M solution of urea.

The reference markers for molecular weight used herein were kits for HMWand LMW gel filtration correction (available from Pharmacia Biotech.).In this connection, the detection was carried out at 254 nm using a UVdetector.

Technical terms concerning the molecular weight determination willhereunder be described:

Molecular Weight: This is a weight average molecular weight or a value(molecular weight) determined by integrating the area surrounded by theelution curve. This is dependent on the whole peptides present in thesample and requires a variety of molecular weights.

Peak Molecular Weight: This is the molecular weight corresponding to thepeak of the elution curve. This corresponds to the molecular weight ofpeptides present in the sample and requires the maximum population. Inthis case, the molecular weight distribution is not taken intoconsideration.

EXAMPLE 1 Preparation of Aqueous Solution of Bombyx mori Silk Fibroinand Bombyx mori Silk Fibroin Membrane

First, cocoon fibers from Bombyx mori silkworm were immersed into amixed aqueous solution containing 0.2% Marcel Soap and 0.05% sodiumcarbonate. The mixture was boiled at 98° C. for 30 minutes to remove thesericin adhering the outer layer of the cocoon fibers and to preparesilk fibroin fibers. The resulting silk fibroin fibers (10 g) wereimmersed in an 8.5M lithium bromide aqueous solution at a temperature ofnot less than 55° C. for 15 minutes to dissolve the silk fibroin fibers.This aqueous neutral salt solution was poured in a dialysis membrane ofcellulose and both ends of the membrane were tied up with sawingthreads. The solution was then dialyzed against tap water maintained at5° C. for 2 days to completely remove the lithium ions and bromide ionspresent therein and to thus give an aqueous solution of pure Bombyx morisilk fibroin. Aqueous solutions of silk fibroin having a variety ofconcentrations were prepared by partially evaporating the water of theresulting silk fibroin aqueous solution or diluting it with water. Theseaqueous solutions were used in the following Examples.

The silk fibroin aqueous solution thus prepared was cast dried on apolyethylene substrate at room temperature to form a Bombyx mori silkfibroin membrane.

EXAMPLE 2 Preparation of Aqueous Solution of Silk Fibroin from Antheraeapernyi and Membrane of Antheraea pernyi Silk Fibroin

First, cocoon fibers from Antheraea pernyi were immersed in a 0.1%aqueous solution of sodium peroxide at 98° C. for one hour to remove thesilk sericin and tannin covering the surface of the cocoon fibers fromAntheraea pernyi and to prepare Antheraea pernyi silk fibroin fibers(material-to-liquor ratio 1:50). The Antheraea pernyi silk fibroinfibers whose sericin and tannin had been removed in advance weredissolved in an aqueous lithium thiocyanate solution. The resultingaqueous solution was poured into a dialysis membrane of cellulose andboth ends of the membrane were tied up with sawing threads. The solutionwas dialyzed against tap water maintained at room temperature for 2 daysto completely remove the lithium ions and thiocyanate ions presenttherein and to give an aqueous solution of pure Antheraea pernyi silkfibroin.

The Antheraea pernyi silk fibroin aqueous solution prepared was castdried on a polyethylene substrate at room temperature to form anAntheraea pernyi silk fibroin membrane.

EXAMPLE 3 Hybrid Membrane of Bombyx mori Silk Fibroin and Antheraeapernyi Silk Fibroin

Predetermined amounts of the aqueous solutions of Bombyx mori silkfibroin and Antheraea pernyi silk fibroin prepared in Examples 1 and 2,respectively, were added to a beaker. These solutions were carefullyadmixed together by gentle stirring with a glass rod in such a mannerthat the aqueous solutions never underwent any gelation (orprecipitation). The mixed aqueous solution thus prepared was cast driedon a polyethylene substrate at room temperature to form a transparenthybrid membrane.

The concentrations of both aqueous solutions of Bombyx mori silk fibroinand Antheraea pernyi silk fibroin were set at a level of 0.1 to 3 wt %.The use of aqueous solutions having a concentration falling within therange would permit the appropriate control of the amounts of theseaqueous solution required for the preparation of such a hybrid membraneand efficient operations. In addition, the use thereof permitted theuniform mixing of these two liquids, and as a result, a hybrid membranehaving uniform quality could be obtained.

EXAMPLE 4 Hybrid Membrane of Bombyx mori Silk Fibroin and Cellulose

First, Bombyx mori silk fibroin fibers and commercially availablepowdery cellulose free of any particular purification (available fromFluka Company) were separately dissolved in a cuprammonium solution([Cu(NH₃)₄](OH)₂) to prepare corresponding aqueous solutions. Then,these two kinds of aqueous solutions were admixed together with gentlestirring such that the Bombyx mori silk fibroin fibers/cellulosecomposition was equal to 80/20, 60/40, 40/60 or 20/80. The mixed aqueoussolution thus prepared was gently cast dried on a glass plate arrangedon a horizontal plane. A mixed solution containing acetone and aceticacid (4:1 (v/v)) was carefully added to the surface of the mixed aqueoussolution to remove the metal complex present in the mixed aqueoussolution while solidifying or coagulating the Bombyx mori silk fibroinand the cellulose. Thereafter, the resulting membrane was washed with amixed solution containing glycerin and water (7:13 (v/v)), then withwater, and then dried at room temperature to give a hybrid membrane ofBombyx mori silk fibroin and cellulose. The resulting membrane had athickness ranging from about 10 to 30:m.

EXAMPLE 5 Hybrid Membrane of Bombyx mori Silk Fibroin and Chitin,Chitosan Derivatives

In this Example, the aqueous solution of Bombyx mori silk fibroinprepared in Example 1 was admixed with an aqueous solution of chitin ora chitosan derivative as a secondary substance to prepare a hybridmembrane of Bombyx mori silk fibroin and chitin or a chitosan derivativeas a secondary substance.

First, powdery chitin was suspended in a 42% aqueous solution of sodiumhydroxide and stirred for 4 hours under reduced pressure. Then, theresulting powdery chitin was charged into a 60% aqueous sodium hydroxidesolution containing sodium dodecyl-sulfate, stirred and then allowed tostand at −20° C. overnight. Alternatively, the resulting powdery chitinwas suspended in liquid ammonia (−33° C.) and then metal potassium wasadded to the suspension to give alkali chitin. The alkali chitin thusprepared was compressed, dispersed in finely divided ice and thensulfurized by the addition of carbon disulfide to thus give chitinsulfide. The aqueous solution of this sulfide was used as an aqueoussolution of a secondary substance.

Separately, the alkali chitin and ethylene chlorohydrin(2-chloroethanol) were reacted under the known reaction conditions togive ethylene glycol chitin. This chitin was treated in a 40% aqueoussodium hydroxide solution at 100° C. for 5 hours with stirring to formwater-soluble glycol chitosan. The glycol chitosan was dissolved in anaqueous acetic acid solution and the resulting solution was used as anaqueous solution of a secondary substance.

Then, to each of the aqueous solution of sulfide and the aqueoussolution of glycol chitosan prepared according to the foregoing methods,there was added an aqueous solution of Bombyx mori silk fibroin preparedaccording to the method used in Example 1 to give each correspondingaqueous mixed solution. This aqueous mixed solution was cast dried on apolyethylene substrate to form a transparent, soft membrane-likecomposite material comprising chitin sulfide or glycol chitosan andBombyx mori silk fibroin.

EXAMPLE 6 Hybrid Membrane of Bombyx mori Silk Fibroin and Wool Keratin

To dissolve wool yarns, Cystine cross linkings thereof were cleavedusing mercapto-ethanol or thioglycollic acid in a nitrogen gasatmosphere to solubilize the keratin molecules through reduction. Whenusing mercapto-ethanol, the reduction was carried out in a urea solutionhaving a concentration of 8M. When using thioglycollic acid, thereduction was carried out by the addition of 4% NaCl.

More specifically, degreased wool yarns were immersed in a urea solutionhaving a concentration of 8M, followed by degassing, addingmercapto-ethanol in an amount of 5 mL per 10 g of wool yarns at atemperature of 25° C. in a nitrogen gas atmosphere and stirring over 3hours to reduce the wool keratin molecules and to give wool keratincarrying SH groups. Then, the resulting wool keratin was dialyzedagainst pure water to remove the urea and the excess mercapto-ethanoland to give an aqueous solution of wool keratin. This aqueous woolkeratin solution was used as an aqueous solution of a 40 secondarysubstance.

Moreover, to 10 g of the reduced wool keratin obtained according to theforegoing method, 15 g of iodoacetic acid at 25° C. was added withstirring in a nitrogen gas atmosphere to react the reduced wool keratinand the iodoacetic acid. After 2 hours, the pH value of the reactionsystem was adjusted to about 8.5. The resulting reaction solution waspoured into a dialysis membrane of cellulose and both ends of themembrane were tied up with sawing threads. The reaction solution wasdialyzed against pure water to remove the excess iodoacetic acid presenttherein and to give an aqueous solution of S-carboxymethyl keratin. Thisaqueous solution was used as an aqueous solution of a secondarysubstance.

To each of the aqueous reduced wool keratin solution and the aqueoussolution of S-carboxymethyl keratin prepared according to the foregoingprocedures, there was added an aqueous solution of Bombyx mori silkfibroin prepared according to the procedures used in Example 1 to giveeach corresponding aqueous mixed solution. This aqueous mixed solutionwas cast dried on a polyethylene substrate to form a hybrid membrane ofreduced keratin or S-carboxymethyl keratin and Bombyx mori silk fibroin.

EXAMPLE 7 Hybrid Membrane of Bombyx mori Silk Fibroin and PolyvinylAlcohol

Polyvinyl alcohol (PVA) was added to hot water of 85° C. and carefullydissolved therein using a stirring machine. The resulting solution wasallowed to stand at room temperature for 30 minutes to give a 0.5%aqueous solution of PVA. To this PVA aqueous solution, there was added a0.3% w/v aqueous solution of Bombyx mori silk fibroin prepared accordingto the procedures used in Example 1. The resulting aqueous mixedsolution was cast dried on a polyethylene substrate over a whole day andnight to form a transparent hybrid membrane of PVA and Bombyx mori silkfibroin.

EXAMPLE 8 Peel Resistance of Silk Fibroin Membrane with Respect to PETSubstrate

A silk fibroin membrane was adhered to the surface of a polyethyleneterephthalate (PET; trade name: Tetoron) membrane according to thefollowing method. The foregoing Tetoron membrane was immersed in a 5%aqueous Bombyx mori silk fibroin (BF) solution prepared according to theprocedures used in Example 1, a 3% aqueous Antheraea pernyi silk fibroin(TF) solution prepared according to the procedures used in Example 2 oran equivalent mixed aqueous solution of the 5% aqueous Bombyx mori silkfibroin (BF) solution and the 3% aqueous Antheraea pernyi silk fibroin(TF) solution, withdrawn therefrom and then dried at room temperature.Each of these membranes (hereunder abbreviated as “PET/BF”, “PET/TF”,“PET/(BF+TF)” respectively) was subjected to the determination of ATR(Attenuated Total Reflection) spectra. This ATR spectroscopic analysiswas carried out using an ATR infrared spectrophotometer (FT-IR5300available from Nippon Bunko K.K.) (Resolution 2; Number of Scanning: 32;gain: 100; using Apdization CS). In addition, a friction element wasreciprocated 10 times on a membrane sample using an abrasion machine.Then, the membrane sample was again subjected to the ATR spectroscopicanalysis. The results observed for the sample prior to the friction testare listed in the following Table 1.

TABLE 1 Sample Wave Number (cm⁻¹) and Absorption Intensity PET 1711(vs), 1408 (s), 1338 (s) PET/BF 1687 (s), 1657 (vs), 1554 (s), 1408 (s),1338 (s), 650 (s) PET/TF 1688 (s), 1655 (vs), 1408 (s), 1338 (s), 620(s) PET/(BF + TF) 1789 (s), 1657 (vs), 1655 (vs), 1408 (s), 1338 (s),650 (w), 620 (s)

In Table 1, symbols “vs, s” and “w” indicate that the spectralintensities are very strong, strong and weak, respectively.

As will be clear from the data listed in Table 1, the ATR spectraobserved for PET/BF include absorption peaks ascribable to PET (1408 and1338 cm⁻¹) and absorption peaks ascribable to Bombyx mori silk fibroin(1657 and 650 cm⁻¹), which are superimposed to one another. Moreover,the ATR spectra observed for PET/TF include absorption peaks ascribableto Antheraea pernyi silk fibroin (1655 and 620 cm⁻¹) in addition tothose ascribable to PET, which are overlapped to one another. The ATRspectra observed for PET/BF and PET/TF obtained after the frictionelement was reciprocated 10 times thereon were identical to thoseobserved for PET prior to the adhesion of these silk fibroin membranes.

The foregoing results clearly indicate that when the PET/BF and PET/TFlaminate surface are treated in an abrasion machine, the BF or TFmembrane on the surface of PET substrate is scraped off during thereciprocating motions of the friction element. In the case of thePET/(BF+TF) laminate, however, the absorption peaks ascribable to BF andthe absorption peaks ascribable to TF still remain in the ATR spectraobserved for the PET/(BF+TF) laminate even after the laminate surface istreated in an abrasion machine. This clearly indicates that BF+TF iscertainly adhered to the surface of the PET substrate and scarcelypeeled off therefrom even when mechanically rubbed.

In addition, a PET substrate having a size of 2 cm H 3 cm was immersedin an aqueous solution containing simply Bombyx mori or Antheraea pernyisilk fibroin and a mixed aqueous solution prepared by admixing anaqueous solution of Bombyx mori silk fibroin and an aqueous solution ofAntheraea pernyi silk fibroin by carefully and gently stirring with aglass rod at room temperature in a desired mixing ratio (at 25° C. for10 minutes). Then, the PET covered with each solution was withdrawn fromeach aqueous solution and cast dried at room temperature. To make thesilk protein membrane on the PET substrate insoluble, the coated PETsubstrate with silk fibroin was immersed in a 50% aqueous solution ofmethanol for 5 minutes, withdrawn therefrom and then dried at roomtemperature. The processed membrane thus prepared was subjected to thesame ATR spectrometric analysis described above in connection with theforegoing membrane free of any insolubilization treatment.

As a result, absorption peaks were observed ascribable to Bombyx morisilk fibroin membrane in addition to those ascribable to the PETsubstrate in the case of the membrane simply consisting of Bombyx morisilk fibroin; absorption peaks ascribable to Antheraea pernyi silkfibroin membrane in addition to those ascribable to the PET substrate inthe case of the membrane simply consisting of Antheraea pernyi silkfibroin; and absorption peaks ascribable to Bombyx mori and Antheraeapernyi silk fibroin membranes in addition to those ascribable to the PETsubstrate in the case of the hybrid membrane. This fact clearlyindicates that the surface of the PET substrate is covered with amembrane consisting of Bombyx mori silk fibroin, Antheraea pernyi silkfibroin or a hybrid thereof.

Then, the peel resistance of various coated membrane with respect to aPET substrate were evaluated according to the foregoing method using PETsubstrates whose surfaces were covered with a membrane of Bombyx morisilk fibroin, a membrane of Antheraea pernyi silk fibroin and a hybridmembrane of Bombyx mori and Antheraea pernyi silk fibroins,respectively.

As a result, it was found that the membrane simply consisting of Bombyxmori or Antheraea pernyi silk fibroin had a tendency to be slightlyeasily peeled off from the PET substrate, while the hybrid membrane hada tendency that it was hardly peeled off therefrom and was excellent inthe adhesion stability. In other words, it was found that theinteraction between the surface of the PET substrate and the hybridmembrane (BF+TF membrane) is higher than those observed between thesurface of the PET substrate and the BF membrane and between the surfaceof the PET substrate and the TF membrane. More specifically, a BF or TFmembrane adhered to a PET substrate is easily peeled off from the PETsubstrate when any mechanical frictional force is applied to eachlaminate, but a hybrid membrane is strongly interacted with a PETsubstrate and therefore, the former is hardly peeled off from thelatter.

EXAMPLE 9 Cell-growth Ability on Hybrid Membrane Surface

The cell-growth behaviors of Bombyx mori cells (Bm cells) and Antheraeapernyi cells (Ae cells) on the membrane surface were examined accordingto the method described above using a Bombyx mori silk fibroin membrane(BF membrane), an Antheraea pernyi silk fibroin membrane (TF membrane)and a hybrid membrane (BF+TF membrane) prepared according to theprocedures used in Examples 1, 2 and 3, respectively. The resultsobtained indicate that both of these cells show almost the same tendencyin the cell-growth behavior. Therefore, only the results observed forthe Bm cells are summarized in the following Table 2.

TABLE 2 Component of Membrane Degree of Cell-Growth BF ± TF ± BF + TF +

In Table 2, “±” means that the degree of cell-growth is almost identicalto or slightly superior to that observed for the polystyrene surface asa control substrate and “+” means that the degree of cell-growthvisually judged is superior to that observed for the polystyrenesurface.

As will be seen from the results listed in Table 2, the degree of insectcell-growth observed on the surface of a culture medium covered with thehybrid membrane of BF and TF is higher than that observed on the surfaceof a culture medium covered with the Bombyx mori silk fibroin membrane(BF membrane) or the wild silkworm silk fibroin membrane (TF membrane).

EXAMPLE 10 Optimum Biodegradation Conditions

In this Example, it is intended to determine the optimum conditions forthe biodegradation test carried out in the following Examples. This isbecause various conditions should be adjusted such that the enzyme usedcan maintain its maximum activity even when changing the kind andconcentration of the enzyme. For instance, the kind of the bufferingsolution and the pH value should appropriately be selected.

The optimum conditions for evaluating the decomposition behavior ofbiodegradable biopolymer material with enzymes are as follows. Theoptimum conditions, such as the kinds of enzymes, enzyme activities andoptimum pH values of buffering solutions, are summarized in thefollowing Table 3. The biodegradation temperature was set at 37° C. Asenzymes, three kinds of enzymes were used, chymotrypsin, collagenase andprotease. The material-to-buffering solution ratio 1:250 was maintainedand the biodegradation behavior was monitored or examined over 570hours. The enzyme concentration was expressed in terms of the mass (mg)of each enzyme per 1 mL of the culture medium. The chymotrypsin, thecollagenase (Type F) and the protease used were all available from SigmaAldrich Japan Co., Ltd.

The buffering solutions used in this Example were TES (N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid available from WakoPure Chemical Industries, Inc.) for the biodegradation tests usingcollagenase;tris(hydroxymethyl)-amino-methane(2-amino-2-hydroxymethyl-1,3-propanediolavailable from Wako Pure Chemical Industries, Inc.) for thebiodegradation tests using chymotrypsin; and potassium phosphatebuffering solution for the biodegradation tests using protease.

TABLE 3 Enzyme Activity Concn. (units/ Enzyme (mg/mL) Buffering Solutionmg solid) pH Collagenase 0.2, 0.5 50 mL TES, 50 mM CaCl₂ 1.8-2.2 7.4Chymotrypsin 0.2, 0.5 50 mM Tris, 5 mM CaCl₂ 40-60 7.8 Protease 0.2, 0.540 mM Potassium 5.7 7.5 phosphate (pH7.5)

EXAMPLE 11

In this Example, the biodegradation behaviors of Bombyx mori silkfibroin membrane or Antheraea pernyi silk fibroin membrane wereinvestigated.

The cocoon layer of Bombyx mori was cut into pieces having a size of ¼time the original one. The waxes and dyestuffs included in the samplewere removed in a Soxhlet extractor using an ethanol/benzene mixedsolution (1:2 v/v). Then, the cocoon fiber sample was charged into amixed solution containing 0.2% Marcel Soap and 0.05% sodium carbonate.The mixture was then boiled at 98° C. for 30 minutes to remove sericinas an adhesive substance present in the outer layer of the cocoon fiber.At this stage, the material-to-liquor ratio was set at 1:100. Ten gramsof Bombyx mori silk fibroin fibers thus prepared were immersed in a 8.5M aqueous solution of lithium bromide at a temperature of not less than55° C. for 15 minutes to solubilize the silk fibers. This aqueousneutral salt solution was poured into a dialysis membrane of celluloseand both ends of the membrane were tied up with sawing threads. Theaqueous solution was then dialyzed against pure water maintained at roomtemperature for 4 days to completely remove the lithium and bromide ionspresent therein and to give an aqueous solution of Bombyx mori silkfibroin having a concentration of 0.2%.

Alternatively, cocoon fibers from Antheraea pernyi were degummed in a0.1% aqueous sodium peroxide solution in an amount of 50 times the massof the cocoon fibers at a temperature of 98° C. for one hour to removesericin and tannin. The Antheraea pernyi silk fibroin fibers from whichsericin and tannin had been removed were dissolved in an aqueoussolution of lithium thiocyanate maintained at 55° C. The resultingaqueous solution was poured into a dialysis membrane of cellulose andboth ends of the membrane were tied up with sawing threads. The aqueoussolution was then dialyzed against pure water to give an aqueoussolution of Antheraea pernyi silk fibroin having a concentration of0.3%.

The 0.2% aqueous solution of Bombyx mori silk fibroin and the 0.3%aqueous solution of Antheraea pernyi silk fibroin prepared according tothe procedures used above were separately cast dried on a polyethylenesubstrate at room temperature to form a Bombyx mori silk fibroinmembrane (BF membrane) and an Antheraea pernyi silk fibroin membrane (TFmembrane), respectively.

The Bombyx mori silk fibroin membrane and the Antheraea pernyi silkfibroin membrane thus prepared were proteolyticaly digested for therelation between the biodegradation behaviors by the action of a varietyof enzymes and the biodegradation time (0, 24, 72, 240, 576 hours). Therates (%) of the residual sample weight as a function of thebiodegradation time are summarized in the following Table 4. The enzymeconcentration was set at 0.2 and 0.5 mg/mL.

TABLE 4 Biodegradation Time (hr.) Sample Enzyme (Concn.) 0 24 72 240 576Bombyx mori Silk Control 100 98 — — — Fibroin Membrane Collagenase (0.2mg/mL) 100 98 95.5 93.5 90 Collagenase (0.5 mg/mL) 100 97.8 96.5 91.087.5 Chymotrypsin (0.5 mg/mL) 100 90.9 90.5 89.7 90.6 Protease (0.2mg/mL) 100 81.8 75.6 71.6 54.5 Protease (0.5 mg/mL) 100 72.7 43.5 44.427.9 Antheraea pernyi Silk Protease (0.2 mg/mL) 100 96.9 88.6 88 86.2Fibroin Membrane Protease (0.5 mg/mL) 100 88.8 89.8 87.2 76.1

The enzyme concentrations listed in Table 4 are expressed in terms ofthe amount (mg) of each enzyme added to the biodegradation medium per 1mL of the latter.

The data listed in Table 4 clearly indicate that the Bombyx mori silkfibroin membrane is susceptible to the digestion with protease and thatin the biodegradation experiment at a protease concentration of 0.2mg/mL, the rate of the residual sample weight is found to be 54.5% afterthe biodegradation time of 576 hours. On the other hand, when acting thesame concentration of protease on the Antheraea pernyi silk fibroinmembrane, the rate of the residual sample weight is found to be 86.2%after the biodegradation time of 576 hours. This indicates that theBombyx mori silk fibroin membrane is more susceptible to the digestionwith protease as compared with the Antheraea pernyi silk fibroinmembrane.

EXAMPLE 12 Crystallinity Index

When the Bombyx mori silk fibroin membrane is biodegraded, the mass ofthe membrane undergoes changes and the digestion reaction thereof isadvanced with the elapse of the biodegradation time. In this Example,the crystallinity index of each membrane was investigated in order toclarify any change in the fine structure of the Bombyx mori silk fibroinmembrane during the biodegradation process.

The Bombyx mori silk fibroin membrane, which had been biodegraded with avariety of enzymes for a predetermined period of time, was evaluated forthe crystallinity index (CI) according to the foregoing method. In thisconnection, CI has no dimension. The results thus obtained are listed inthe following Table 5.

TABLE 5 Biodegradation Time (hr.) Enzyme (Concn.) 0 72 240 408Collagenase (0.2 mg/mL) 0.547 0.55 0.56 0.568 Chymotrypsin (0.2 mg/mL)0.55 0.557 0.568 0.569 Protease (0.2 mg/mL) 0.547 0.585 0.594 0.601Protease (0.5 mg/mL) 0.547 0.607 0.617 0.615

As will be clear from the data listed in Table 5, the amorphous regionof the Bombyx mori silk fibroin membrane is digested through thedigestion reaction with the enzyme and, as a result, the crystallineregion thereof increases.

EXAMPLE 13 Biodegradation Behavior of Silk Fibers

The biodegradation behaviors of Bombyx mori silk fibers and Antheraeapernyi silk fibers were investigated on the basis of the rate of theresidual sample weight according to the same procedures used in Example11. The results obtained are summarized in the following Table 6.

TABLE 6 Biodegradation Time (hr.) Sample Enzyme (Concn.) 0 24 72 240 576Bombyx mori Silk Control 100 100 — — — Fiber Collagenase (0.2 mg/mL) 100100 100 100 99 Collagenase (0.5 mg/mL) 100 99.5 99 99 98 Bombyx moriSilk Control 100 99 — — — Fiber Chymotrypsin (0.2 mg/mL) 100 99.5 99.599.3 99 Chymotrypsin (0.5 mg/mL) 100 99 99 99 99 Bombyx mori SilkControl 100 100 — — — Fiber Protease (0.2 mg/mL) 100 98.6 99.3 99.3 99.3Protease (0.5 mg/mL) 100 100 100 99.8 99.3 Antheraea pernyi Control 100100 — — — Silk Fiber Collagenase (0.2 mg/mL) 100 100 100 98 97Collagenase (0.5 mg/mL) 100 100 100 100 99 Antheraea pernyi Control 100100 — — — Silk Fiber Chymotrypsin (0.2 mg/mL) 100 100 99 99 98Chymotrypsin (0.5 mg/mL) 100 100 100 100 100 Antheraea pernyi Control100 100 — — — Silk Fiber Protease (0.2 mg/mL) 100 100 100 99.9 99.5Protease (0.5 mg/mL) 100 99.2 99.0 98.4 99.2

The results listed in Table 6 indicate that the Bombyx mori silk fiberand the 15 Antheraea pernyi silk fiber are scarcely biodegraded on thebasis of the decreasing rate of the residual sample weight. However, thesilk fiber may undergo changes in fine structures and may sufferdeterioration in addition to weight changes during the biodegradationprocess. For this reason, the Bombyx mori silk fiber was evaluated forthe deterioration during the biodegradation process on the basis of thestrength and elongation according to the following method.

Bombyx mori silk fibers were added to a culture medium containingprotease, collagenase or chymotrypsin. As such, they were biodegradedover a predetermined time period and then the changes with time of thestrength and elongation at break of the silk fibers after thebiodegradation were determined. The results thus obtained are summarizedin the following Table 7.

TABLE 7 Biodegradation Time (hr.) 0 24 72 240 408 Strength (N)Collagenase (0.2 mg/mL) 4.68 3.60 4.12 3.86 3.64 Chymotrypsin (0.2mg/mL) 4.68 3.83 3.72 3.97 3.78 Protease (0.2 mg/mL) 4.68 3.74 3.52 —3.14 Protease (0.5 mg/mL) 4.68 3.83 3.77 — 3.23 Elongation (%)Collagenase (0.2 mg/mL) 32.7 26.3 25.1 25.7 23.5 Chymotrypsin (0.2mg/mL) 32.7 28.1 27.8 26.4 25.8 Protease (0.2 mg/mL) 32.7 21.6 20.5 —18.1 Protease (0.5 mg/mL) 32.7 24.1 23.2 — 19.0

In Table 7, the enzyme concentration is given in parentheses behind eachcorresponding enzyme name and it is expressed in terms of the amount ofthe enzyme (mg) per 1 mL of the culture medium. Moreover, the strengthof the silk fiber expressed in N can be converted into that in kg on thebasis of the equation: (numerical value of each strength)/9.81. In thisTable, for instance, 4.68 N corresponds to 477.1 g.

The data listed in Table 7 clearly indicates that both the strength andthe elongation of the silk fiber decrease with an increase ofbiodegradation time. The data listed in Table 6 indicates that the silkfiber is not biodegraded at first view, but it is clear that thedeterioration of the silk fiber is in fact advanced due to thebiodegradation.

EXAMPLE 14 Biodegradability of Hybrid Membrane

A hybrid membrane sample prepared from Bombyx mori silk fibroin (BF) andAntheraea pernyi silk fibroin (TF) according to the procedures used inExample 3 and a hybrid membrane prepared from Bombyx mori silk fibroin(BF) and cellulose (Cell) according to the procedures used in Example 4were examined for the relation between the biodegradation time and therate (%) of the residual sample weight observed when they are hydrolyzedwith protease. The results thus obtained are listed in the followingTable 8.

TABLE 8 Biodegradation Time (hr.) Sample Presence of Enzyme 24 72 240408 572 BF:TF (8:2) present 85.4 81.8 77.3 75.0 75.0 BF:TF (8:2) absent100 100 100 100 100 BF:TF (6:4) present 85 84.2 84.2 84.2 78.9 BF:TF(6:4) absent 95.4 95.4 94.4 100 100 BF:TF (4:6) present 100 91.3 86.986.4 86.4 BF:TF (4:6) absent 100 100 100 100 95.5 BF:TF (2:8) present100 100 96.9 97.1 96.9 BF:TF (2:8) absent 100 100 100 100 95.5 BF:Cell(8:2) present 86.4 87.9 51.7 50 52.1 BF:Cell (8:2) absent 88.8 89.5 89.590 90 BF:Cell (6:4) present 77.3 72 65.4 60.8 61.5 BF:Cell (6:4) absent94.1 96.7 97.1 100 96.9 BF:Cell (4:6) present 78.6 75 73.0 73.3 73.3BF:Cell (4:6) absent 100 100 100 100 100 BF:Cell (2:8) present 94.1 94.194.1 87.5 82.4 BF:Cell (2:8) absent 94.4 100 100 100 100 BF:Cell (0:10)present 97.5 94.1 100 97.1 97.7 BF:Cell (0:10) absent 93.7 95.7 96.797.7 96.3

In the column entitled “Presence of Enzyme” in Table 8, each sectionspecified by “absent” means the biodegradation experiment, which isconducted in an aqueous solution containing only a buffering solutionand free of any proteolytic enzyme, while, each section specified by“present” means the biodegradation experiment, which is carried out inan enzyme-containing decomposition solution containing both protease anda buffering solution. In addition, “BF:TF (4:6)” means a hybrid membraneprepared by admixing an aqueous solution of Bombyx mori silk fibroin andan aqueous solution of Antheraea pernyi silk fibroin, such that theresulting mixed solution contains 40% Bombyx mori silk fibroin and 60%Antheraea pernyi silk fibroin, and then cast drying. “BF: Cell (8:2)”means a hybrid membrane prepared in such a manner that the resultinghybrid membrane comprises 80% Bombyx mori silk fibroin and 20%cellulose.

As will be seen from the data listed in Table 8, in the case of thehybrid membrane of Bombyx mori silk fibroin and Antheraea pernyi silkfibroin (BF: TF membrane) the hybrid membrane is hardly biodegradable asa whole, as the content of the Antheraea pernyi silk fibroin increases.Moreover, in the case of the hybrid membrane of Bombyx mori silk fibroinand cellulose (BF: Cell membrane), the hybrid membrane is likewisehardly biodegradable.

EXAMPLE 15 Hybrid Membrane of Bombyx mori Silk Fibroin and Wool Keratin

An aqueous solution of wool keratin was prepared as follows.

Pigments and greases contained in wool (64′ S) from sheep belonging tothe Merino species were removed by treating the same with abenzene/ethanol (50/50 (% by volume)) mixed solvent for 2.5 hours usinga Soxhlet extractor.

A three-necked flask was used. The first neck thereof was connected toone end of a rubber tube, the other end of which was connected to anitrogen gas cylinder for drying through a three-way cock. The secondneck thereof was always occupied by a pH electrode assembly for thecontrol of the pH value of the reaction system. The third or remainingneck or port was used for the introduction of any necessary reagent intothe system. Wool yarns (8.18 g) from sheep of Merino species, which hadbeen cut into short yarns having a yarn length of about 1 cm, werecharged into the three-necked flask. Then, 450 mL of an 8M aqueous ureasolution was added to the flask. The flask was purged with nitrogen gas,the pressure in the flask was reduced to about 45 mmHg for 15 minutesusing an aspirator and then the pressure in the flask was abruptlyreturned to the atmospheric pressure, these operations being repeatedthree to four times. Thus, the air contained in the wool yarns presentin the three-necked flask was completely removed so that the reaction ofthe aqueous urea solution with keratin molecules would be efficientlyadvanced. After the completion of the displacement with nitrogen gas,4.8 mL of mercapto-ethanol as a reducing agent was added to thethree-necked flask and the wool yarns were allowed to stand in the 8Maqueous urea solution over 2 to 3 hours. Then, about 100 mL of a 5N-KOHaqueous solution was added to the flask in small portions to thus adjustthe pH value of the mixed aqueous solution in the flask to 10.5. Thecontent of the flask was allowed to stand at room temperature for 3hours until the wool yarns were completely dissolved in the aqueoussolution to give an aqueous keratin solution. The resulting aqueouskeratin solution was poured into a dialysis membrane of cellulose andboth ends of the membrane were tied up with sawing threads. The aqueouskeratin solution was then dialyzed against pure water for 2 days. Theresulting aqueous keratin solution was subjected to drying throughventilation or, if desired, diluted with pure water to give an aqueouskeratin solution having a desired keratin concentration.

The keratin in the 0.01% aqueous keratin solution thus prepared wassubjected to an S-carboxy-methylation reaction at room temperature forone hour by the addition of 9.5 g of iodoacetic acid to 450 mL of theaqueous keratin solution. The pH value of the aqueous keratin solutionwas adjusted to 8.5 by the addition of 5N-KOH aqueous solution to givean aqueous solution of S-carboxymethyl keratin solution. This aqueoussolution was poured into a dialysis membrane of cellulose and both endsof the membrane were tied up with sewing yarns. The aqueous solution wasthen dialyzed against pure water for 2 days.

EXAMPLE 16 Weight-Average Molecular Weight of Bombyx mori Silk FibroinMembrane and Bombyx mori Silk Fiber

A Bombyx mori silk fibroin membrane and Bombyx mori silk fibers wereenzymatically decomposed, sufficiently washed with water and then dried.The resulting sample was subjected to HPLC measurements to determine theweight-average molecular weight thereof. The results thus obtained arelisted in the following Table 9.

TABLE 9 Weight-Average Molecular Weight (kD) Enzyme Processing Time(hr.) Sample (Concn: mg/mL) 0 72 240 408 576 Bombyx mori Collagenase(0.2) 119.8 98.5 96.8 94.3 — Silk Fibroin Chymotrypsin (0.2) 119.8 77.465.7 53.7 — Membrane Protease (0.2) 119.8 109.6 105.6 102.4 — Bombyxmori Collagenase (0.2) 233.1 184.3 — — 204.4 Silk Fiber Protease (0.2)233.1 256.5 — — 247.4 Protease (0.5) 233.1 261.7 — — 241.7

In Table 9, each numerical value given in parentheses appearing in thecolumn entitled “Enzyme” means the concentration of an enzyme used. Thenumerical value “0.2” means that the culture medium contains 0.2 mg ofthe enzyme per 1 mL of the medium.

As will be clear from the data listed in Table 9, the molecular weightof the Bombyx mori silk fibroin membrane was found to be graduallyreduced from its initial value of 120,000 D after the proteolyticreaction with the enzyme. More specifically, it was confirmed that theweight-average molecular weight of the membrane was reduced to about94,000 D and 100,000 D after 408 hours from the initiation of thebiodegradation with collagenase and protease, respectively and that theweight average molecular weight of the membrane was rapidly reduced fromits initial value of 120,000 D to about 54,000 D at the samebiodegradation time. The molecular weight of the untreated Bombyx morisilk fiber is about 230,000 D. In this respect, it was found that theweight-average molecular weight thereof was only slightly reduced evenafter the enzymatic digestion.

EXAMPLE 17 Changes in Weight-average Molecular Weight Associated withBiodegradation Treatment

After hydrolyzing the Bombyx mori silk fibroin membrane preparedaccording to the same procedures used in Example 1 with three kinds ofenzymes, collagenase, chymotrypsin and protease, for a predeterminedperiod of time (72, 240 or 408 hours), the membrane was sufficientlywashed with water to give a sample. Each sample obtained after thebiodegradation over a predetermined time was evaluated for theweight-average molecular weight and peak molecular weight. In thisrespect, the weight-average molecular weight thereof was determined bythe high performance liquid chromatography (HPLC) technique. The resultsthus obtained are summarized in the following Table 10.

TABLE 10 Weight-Average Peak Molecular Molecular Weight Enzyme Weight(kD) (kD) Control 119.8 79.8 Collagenase: 0.2 mg/mL  (72 hrs.) 98.5 53.9(240 hrs.) 96.8 52.9 (408 hrs.) 94.3 45.8 Chymotrypsin: 0.2 mg/mL  (72hrs.) 77.4 41.1 (240 hrs.) 65.7 37.6 (408 hrs.) 53.7 34.8 Protease: 0.2mg/mL  (72 hrs.) 109.6 36.4 (240 hrs.) 105.6 33.4 (408 hrs.) 102.4 31.4

In Table 10, the time given in parentheses appearing in the columnentitled “Enzyme” means the elapsed biodegradation time.

The data listed in Table 10 clearly indicate that when an enzyme digestson the Bombyx mori silk fibroin membrane, the weight-average molecularweight of the membrane is reduced from the initial level of 120,000 Dwith the progress of the biodegradation time, but after 72 hours fromthe initiation of the biodegradation, the rate of the molecular weightchange of the membrane was reduced. In particular, in the case ofcollagenase and chymotrypsin, the weight-average molecular weight andthe peak molecular weight were reduced with the progress of thebiodegradation time. On the other hand, in the case of protease, theweight-average molecular weight was reduced with the progress of thebiodegradation time, but the extent of the molecular weight reductionwas insignificant and the peak molecular weight was reduced to an extentalmost identical to that observed for the chymotrypsin.

COMPARATIVE EXAMPLE 1 Molecular Weight Change of Bombyx mori Silk FiberAssociated with Biodegradation Treatment

The changes in the molecular weight of a Bombyx mori silk fiber samplewith the elapsed biodegradation time when digesting the silk fiber withcollagenase, chymotrypsin and protease were monitored and evaluated bythe high performance liquid chromatography technique. The results thusobtained are listed in the following Table 11.

TABLE 11 Weight-Average Peak Molecular Molecular Weight Enzyme Weight(kD) (kD) Control 233.1 179.3 Collagenase: 0.2 mg/mL  (72 hrs.) 184.393.7 (576 hrs.) 204.4 109.7 Chymotrypsin: 0.2 mg/mL  (72 hrs.) 129.466.0 (576 hrs.) 174.4 92.8 Protease: 0.2 mg/mL  (72 hrs.) 256.5 176.9(576 hrs.) 247.4 155.2 Protease: 0.5 mg/mL  (72 hrs.) 261.7 173.5 (576hrs.) 241.7 148.7

In Table 11, the time given in parentheses appearing in the columnentitled “Enzyme” means the elapsed biodegradation time.

The data listed in Table 11 clearly indicates that the Bombyx mori silkfiber hardly causes any reduction of the weight-average molecular weightand the peak molecular weight even when an enzyme acts on the thread, ascompared with the Bombyx mori silk fibroin membrane (Example 17). Forthis reason, the silk fiber is not useful in the fields whereinbiodegradability is required. Materials excellent in biodegradabilityare, for instance, Bombyx mori silk fibroin membranes and Antheraeapernyi silk fibroin membranes.

EXAMPLE 18 Biodegradation Rate

The following samples were digested for the biodegradation rate observedwhen these samples were hydrolyzed with protease, collagenase orchymotrypsin: the Bombyx mori silk fiber and the Antheraea pernyi silkfiber; the Bombyx mori silk fibroin membrane (BF membrane) preparedaccording to the procedures used in Example 1; the Antheraea pernyi silkfibroin membrane (TF membrane) prepared according to the procedures usedin Example 2; the hybrid membrane (BF+TF membrane) consisting of Bombyxmori silk fibroin/Antheraea pernyi silk fibroin prepared according tothe procedures used in Example 3; the hybrid membrane (BF+Cell membrane)consisting of Bombyx mori silk fibroin/cellulose prepared according tothe procedures used in Example 4; and the hybrid membrane (BF+CMKmembrane) consisting of Bombyx mori silk fibroin/carboxymethyl chitinprepared according to the method described above. The results thusobtained are summarized in the following Table 12. In this connection,the term “biodegradation rate” is herein defined to be a value obtainedby dividing the sample weight observed after 50 hours from theinitiation of the biodegradation by the original sample weight (thesample weight prior to the biodegradation) and expressed in terms of“%”. Accordingly, when a sample is not biodegraded at all, thebiodegradation rate of the sample corresponds to 0%/50 hours.

TABLE 12 Enzyme Biodegradation Concn. Rate Sample Enzyme (mg/mL) (%/50hrs.) Bombyx mori Silk fiber Protease 0.2 1.4 ″ ″ 0.4 0 Bombyx mori Silkfiber Collagenase 0.2 0 ″ ″ 0.5 0.5 Bombyx mori Silk fiber Chymotrypsin0.2 0.5 ″ ″ 0.5 1.0 Antheraea pernyi Silk fiber Protease 0.2 0 ″ ″ 0.5 0Antheraea pernyi Silk fiber Collagenase 0.2 0 ″ ″ 0.5 0 Antheraea pernyiSilk fiber Chymotrypsin 0.2 0 ″ ″ 0.5 0 BF Membrane Protease 0.2 18.2 ″″ 0.5 27.3 BF Membrane Collagenase 0.2 2.0 ″ ″ 0.5 2.2 BF MembraneChymotrypsin 0.5 9.1 TF Membrane Protease 0.2 3.1 ″ ″ 0.5 11.2 TFMembrane Chymotrypsin 0.2 14.5 BF:TF (10:0) Protease 0.2 18.2 BF:TF(8:2) ″ 0.2 15 BF:TF (6:4) ″ 0.2 15 BF:TF (4:6) ″ 0.2 0 BF:TF (2:8) ″0.2 0 BF:TF (0:10) ″ 0.2 3.1 BF:Cell (10:0) Protease 0.2 18.2 BF:Cell(8:2) ″ 02 13.6 BF:Cell (6:4) ″ 0.2 22.7 BF:Cell (4:6) ″ 0.2 21.4BF:Cell (2:8) ″ 0.2 5.9 BF:Cell (0:10) ″ 0.2 2.5 BF:CMK (6:4) Protease0.2 12.1 BF:CMK (2:8) ″ 0.2 8.5

In Table 12, “BF:TF” and “BF:Cell” represent the hybrid membrane ofBombyx mori silk fibroin (BF) and Antheraea pernyi silk fibroin (TF) andthe hybrid membrane of Bombyx mori silk fibroin (BF) and cellulose(Cell), respectively and the corresponding numerical values given inparentheses mean that the mixing ratios of BF to TF and those of BF toCell are 100/0, 80/20, 60/40, 40/60, 20/80 and 0/100.

The data listed in Table 12 also clearly indicates that when digesting ahybrid membrane of Bombyx mori silk fibroin and Antheraea pernyi silkfibroin with protease, the hybrid membrane containing Antheraea pernyisilk fibroin in an amount ranging from 60 to 80% does not substantiallyundergo any biodegradation from the viewpoint of the biodegradation rateand this is substantially consistent with the conclusion deduced fromthe data concerning the rate of residual sample weight discussed inExample 13. This is a property, which is never observed for the membranesimply consisting of Bombyx mori silk fibroin or Antheraea pernyi silkfibroin.

EXAMPLE 19 Composite Materials having Different Shapes

A mixture of an aqueous solution of Bombyx mori silk fibroin and anaqueous solution of Antheraea pernyi silk fibroin was poured into abeaker. A dilute aqueous acetic acid solution was gradually added to themixed aqueous solution in small portions to control the pH value of thewhole aqueous solution to 2.5 and to form a gel-like product of Bombyxmori silk fibroin and Antheraea pernyi silk fibroin. Moreover, thegel-like product obtained was directly subjected to lyophilizationwithout removing any moisture from the product according to a knownmethod to prepare a porous product of Bombyx mori silk fibroin andAntheraea pernyi silk fibroin.

Alternatively, a mixture of an aqueous solution of Bombyx mori silkfibroin and an aqueous solution of Antheraea pernyi silk fibroin wascast dried on a polyethylene substrate to give a transparent compositemembrane.

Moreover, acetic acid was added to a mixture of an aqueous solution ofBombyx mori silk fibroin and an aqueous solution of Antheraea pernyisilk fibroin, followed by the control of the pH value thereof to 3.0 andthe lyophilization of the aqueous mixture by a known method to form apowdery hybrid consisting of Bombyx mori silk fibroin and Antheraeapernyi silk fibroin.

EXAMPLE 20 Amino Acid Analysis

A Bombyx mori silk fibroin membrane was digested with protease and thenthe biodegraded sample was subjected to amino acid analysis to examinethe relationship between the results of the amino acid analysis and thebiodegradation time. The results thus obtained are summarized in thefollowing Table 13. In Table 13, the amount of each amino acid residueis expressed in terms of “mole %”.

TABLE 13 Biodegradation Time (day) Amino Acid 0 3 10 17 Cyst 0.03 0.040.02 0.00 Asp 1.28 0.58 0.83 0.59 Glu 1.09 0.54 0.77 0.51 Ser 10.89 9.9610.47 10.68 Gly 45.00 46.49 45.95 46.73 His 0.15 0.10 0.10 0.10 Arg 0.480.32 0.36 0.29 Thr 0.78 0.50 0.59 0.54 Ala 29.43 31.52 30.36 31.71 Pro0.35 0.27 0.28 0.26 Tyr 5.76 5.74 5.80 5.05 Val 2.31 2.17 2.24 1.85 Met0.09 0.07 0.06 0.27 Cyst 0.02 0.00 0.00 0.00 Ile 0.66 0.46 0.59 0.41 Leu0.51 0.31 0.53 0.28 Phe 0.81 0.71 0.77 0.56 Lys 0.36 0.22 0.28 0.17Total 100.00 100.00 100.00 100.00 Gly 45.00 45.95 46.49 46.73 Ala 29.4330.36 31.52 31.71 Ser 10.89 10.47 9.96 10.68 Total-1 85.32 86.78 87.9789.12 Tyr 5.76 5.80 5.74 5.05 Acid 2.40 1.62 1.16 1.10 Basic 0.99 0.740.64 0.56 Others 5.53 5.06 4.49 4.18 Total-2 14.68 13.22 12.03 10.88

In Table 13, “Total” means the amount of the whole amino acids residuesobtained in the amino acid analysis expressed in the unit of mole %,“Total-1” means the total amount of the amino acid residues or Gly, Alaand Ser constituting the crystalline region, while “Total-2” means thetotal amount of acidic amino acid residues (acid) such as Glu and Asp,basic amino acid residues (basic), such as Lys, Arg and His, as well asother amino acids.

As will be seen from the results listed in Table 13, there is observed adistinct tendency in the results obtained by the amino acid analysis ofthe biodegraded Bombyx mori silk fibroin membrane. More specifically,the total amount of Gly, Ala and Ser, which are principal amino acidsconstituting the crystalline region of the silk fibroin, graduallyincreases with the lapse of the digestion time. Meanwhile, the totalamount of bulky polar side chain-containing amino acids, such as Tyr,acidic amino acids and basic amino acids is reduced. In other words, theamino acid composition of the silk fibroin is shifted to that observedfor the crystalline region as the biodegradation proceeds, as describedabove. Therefore, it would be considered that when an enzyme acts onsilk fibroin, the enzyme first acts on the amorphous region susceptibleto the enzymatic attack to induce the digestion of the fibroin. Thecrystalline region of the fibroin almost susceptible to the attack ofthe enzyme remains even after the biodegradation. Accordingly, theprincipal chemical structure of the fibroin is shifted to mainlycomprising crystalline amino acids as the biodegradation proceeds.

EXAMPLE 21 Adsorption of Metal Ions

Experiments were conducted according to the method for adsorbing metalions, as described above. The metal salt aqueous solutions used hereinwere a 0.5 mM aqueous solution of silver nitrate (AgNO₃) and a 0.5 mMaqueous solution of copper nitrate (Cu(NO₃)₂). In each of these aqueoussolutions, a Bombyx mori silk fibroin membrane (BF membrane) preparedaccording to the same procedures used in Example 1, an Antheraea pernyisilk fibroin membrane (TF membrane) prepared according to the sameprocedures used in Example 2 and composite membranes (80:20 and 50:50(weight ratio) BF+TF membranes) consisting of Bombyx mori silk fibroinand Antheraea pernyi silk fibroin prepared according to the sameprocedures used in Example 3, were immersed at a temperature of 25° C.for 30 minutes to adsorb silver ions or copper ions on each sample andto thus determine the quantity of metal ions adsorbed on each sample.

Moreover, various kinds of biodegradable biopolymer membranes on whichsilver ions had been adsorbed according to the foregoing method wereevaluated for the antibacterial activity against tomato canker causalbacterium: Corynebacterium michiganense pv. michiganense.

The amount of the metal ion adsorption and the antibacterial activitythus obtained are listed in the following Table 14.

TABLE 14 Antibacterial Amt. of Activity Sample Amt. of Ag⁺ (mmol/g) Cu²⁺(mmol/g) (mm) BF 0.18 0.23 2 TF 0.24 0.30 2.5 BF + TF (80:20) 0.72 0.958 BF + TF (50:50) 0.93 1.25 8.7

The data listed in Table 14 clearly indicates that the compositemembrane obtained by combining Bombyx mori silk fibroin and Antheraeapernyi silk fibroin may absorb metal ions in an amount greater than thatobserved for the membrane simply consisting of Bombyx mori silk fibroinor Antheraea pernyi silk fibroin. As a result, the former displaysantibacterial activity toward plant pathogenic fungi or bacteria. Thus,the silk fibroin-containing composite material can be used as a metaladsorbent and an antibacterial material.

EXAMPLE 22 Rate of Light Transmission of Silk Protein Membrane

A Bombyx mori silk fibroin membrane (BF membrane), an Antheraea pernyisilk fibroin membrane (TF membrane) and composite membranes (80:20 and50:50 (weight ratio) BF+TF membranes) obtained by hybridizing Bombyxmori silk fibroin and Antheraea pernyi silk fibroin were evaluated forthe transmittance spectra using a self-recording (or autographic)spectrophotometer (Model: W-2100S) available from Shimadzu Corporation.In this connection, the spectra determined were transmittance spectra,including those originated from surface reflection. The resulting ratesof light transmission are listed in the following Table 15.

TABLE 15 Sample Rate of Light Transmission (%) BF Membrane 87.3 TFMembrane 84.7 BF:TF (80:20) Membrane 93.4 BF:TF (50:50) Membrane 90.1

The data listed in Table 15 clearly indicates that the compositemembrane is highly permeable to light rays as compared with the membranesimply consisting of Bombyx mori silk fibroin or Antheraea pernyi silkfibroin or the former has a degree of clearness higher than thatobserved for the latter.

EXAMPLE 23 Drug-sustained Release Properties

To an equivalent mixture of a 2% aqueous solution of Bombyx mori silkfibroin and a 2% aqueous solution of Antheraea pernyi silk fibroinprepared according to the same procedures used in Example 3, there wasgently added an aqueous solution prepared by dissolving 5 mg of acetylsalicylic acid in 50 mL of water. The aqueous mixed solution allowed tostand at room temperature was gradually converted into a gel. Theresulting gel was once frozen at a temperature of −10° C. and then driedin vacuo to form a porous composite material containing acetyl salicylicacid. The porous composite material was digested in an enzyme solutioncontaining 2 mg/mL of protease over a predetermined period of time (24and 72 hours). The amount of the agent gradually released from theporous composite material during the digestion process was evaluated onthe basis of the UV absorbance at 206.9 nm, which was determined using aUV absorbance meter available from Shimadzu Corporation. The resultsthus obtained are listed in the following Table 16. Each UV absorbancevalue listed in Table 16 is obtained by subtracting the UV absorptionobserved for the initial enzyme solution or observed at thebiodegradation time of 0 from the UV absorption observed for the enzymesolution containing the agent gradually released from the porouscomposite material.

Moreover, as a control, a porous material simply consisting of Bombyxmori silk fibroin (BF) or Antheraea pernyi silk fibroin (TF) wasevaluated for the sustained release characteristics according to thesame procedures used above in connection with the foregoing porouscomposite material. The results are summarized in the following Table16.

TABLE 16 Biodegradation Time (hr.) Sample 0 24 72 BF Porous Material0.10 0.259 0.259 TF Porous Material 0.10 0.270 0.271 Porous CompositeMaterial 0.10 0.265 0.293

The data listed in Table 16 clearly indicates that the porous compositematerial containing acetyl salicylic acid gradually releases the drugover a long period of time, as compared with the drug-release behaviorof the porous material simply consisting of Bombyx mori silk fibroin orAntheraea pernyi silk fibroin. This clearly indicates that the compositemembrane possesses drug-sustained release characteristics.

EXAMPLE 24 FT-IR of Composite Membrane

The following membranes were subjected to the FT-IR measurements: aBombyx mori silk fibroin membrane (BF membrane) prepared according tothe procedures used in Example 1; an Antheraea pernyi silk fibroinmembrane (TF membrane) prepared according to the procedures used inExample 2; a composite membrane (BF+TF membrane) consisting of Bombyxmori silk fibroin and Antheraea pernyi silk fibroin prepared accordingto the procedures used in Example 3; a composite membrane (BF+CMKmembrane) consisting of Bombyx mori silk fibroin and carboxymethylchitin used in Example 18; a composite membrane (TF+CMK membrane)consisting of Antheraea pernyi silk fibroin and carboxymethyl chitin; amembrane consisting of CMK alone; and a composite membrane (BF+PVAmembrane) consisting of Bombyx mori silk fibroin and polyvinyl alcohol,to determine wave numbers appearing within the range of from 2000 to 500cm⁻¹. The results thus obtained are summarized in the following Table17.

TABLE 17 Wave Number (cm⁻¹) Sample and Absorption Intensity BF Membrane1654, 1539, 1455, 1414, 1383, 1334, 1240, 1170, 1071, 1061, 1016, 950,670, 532 TF Membrane 1650, 1546, 1274, 615 BF + TF Membrane 1654, 1650,1274, 950, 670, 615, 533 BF + CMK 1654, 1542, 1528, 1451, 1412, 1381,1333, 1242, 1162, 1109, 1070, 949, 666, 559 TF + CMK (2:8) Membrane1657, 1548, 1451, 1378, 1316, 1156, 1113, 1069, 1037, 951, 900, 618, 526TF + CMK (8:2) Membrane 1653, 1542, 1282, 1334, 1307, 659, 617, 525 CMKMembrane 1654, 1592, 1570, 1413, 1374, 1318, 1155, 1111, 1071, 1038,946, 901, 685, 615, 571 BF + PVA (2:8) Membrane 1420-1440, 1326, 1232,1093, 913, 849

In Table 17, the term “TF+CMK (2:8)” means a composite membrane preparedby blending Antheraea pernyi silk fibroin and carboxymethyl chitin in aweight ratio of 20:80. In addition, the term “BF+PVA (2:8)” means acomposite membrane prepared by blending Bombyx mori silk fibroin andpolyvinyl alcohol in a weight ratio of 20:80.

The data listed in Table 17 indicates that the IR spectra observed forthe composite materials consisting of Bombyx mori silk fibroin andsecondary substances, and the composite materials consisting of wildsilkworm silk fibroin and secondary substances, include only spectraascribable to two kinds of constituents, which are superimposed to oneanother and are free of any spectrum other than those ascribable to thetwo constituents. This clearly indicates or suggests that any newlinkage is not formed between the Bombyx mori silk fibroin or the wildsilkworm silk fibroin and the secondary substance.

The foregoing indicates that in the composite materials consisting ofdomesticated or wild silkworm silk fibroin and secondary substancesselected from the group consisting of cellulose, chitin, chitosan,chitosan derivatives, wool keratin and polyvinyl alcohol, there is nochemical or covalent bond between the domesticated or wild silkworm silkfibroin and the secondary substance, but that these two components aresimply coagulated through the action of hydrogen bonds formed inbetween. This is because the composite material is simply prepared, bycasting a mixture of aqueous solutions of respective constituents on thesurface of a substrate and then solidification through evaporation todryness, without using any particular agent for cross-linking molecules.In this connection, the aqueous mixture is allowed to stand and, ifdesired, gently stirred so as not to cause any coagulation of these twokinds of molecules or solidification due to any abrupt mixing mechanicaloperation prior to the casting on the substrate surface.

As discussed above in detail, the biodegradable biopolymer material ofthe present invention comprises an insect's biopolymer alone, such asdomesticated silkworm silk fibroin or wild silkworm silk fibroin, or acomposite material comprising domesticated or wild silkworm silk fibroinand a secondary substance or at least one compound selected from thegroup consisting of cellulose, wool keratin, chitin, chitosan, chitosanderivatives and polyvinyl alcohol, and the biodegradation of thesematerials may be controlled.

The biodegradable biopolymer material of the present invention should beinsolibilized in water prior to the biodegradation experiments, but itis also possible to use any conventionally known agent for cross-linkingprotein molecules, such as formaldehyde or epoxy compounds. In addition,the silk protein membrane or the composite material may be insolubilizedin water by simple treatments, for instance, by lightly immersing themembrane in an aqueous alcohol solution, such as an aqueous methanol orethanol solution, and then drying at room temperature.

The susceptibility of a hybrid to biodegradation may be determined bythe degree of insolubilization of a domesticated or wild silkworm silkfibroin membrane, the choice of the secondary substance, the mixingratio of the domesticated or wild silkworm silk fibroin to the secondarysubstance, the kind of enzyme selected, the enzyme concentration usedand the processing time. Therefore, a hybrid having a desiredbiodegradability can be prepared by appropriately selecting theconditions for producing the same, the mixing ratio of the constituentsand/or the conditions for biodegradation.

The composite material made of domesticated or wild silkworm silkfibroin with secondary substances would permit the achievement of such asignificant effect that the surface of the resulting blending showsexcellent biochemical characteristics, which are never observed for thesurface of the membrane comprising domesticated or wild silkworm silkfibroin alone, or the secondary substance alone. For instance, thesurface of the hybrid is excellent in the rate of biological cell-growthas compared with the surface of the membrane comprising the domesticatedsilkworm silk fibroin alone or the secondary substance alone. Inaddition, the hybrid is also excellent in the ability of coating thesurface of general organic polymers, such as PET, and the use of ahybrid material would permit the improvement of the resistance of amembrane to mechanical friction.

If a useful substance, such as a water-soluble medicine or apharmacological component, is included in the biodegradable biopolymermaterial of the present invention, the medicine or the pharmacologicalcomponent can gradually be released while biodegrading the biodegradablebiopolymer material in the living body. Therefore, the material can beused as a sustained release material.

The biodegradability can be reduced by the use of the silk fibroinfibers from domesticated or wild silkworms. If an easily biodegradablematerial is desired, a membrane-like material may be used. Such amembrane may be prepared by dissolving domesticated or wild silkwormsilk fibers in a neutral salt solution, desalting the resulting solutionusing a dialysis membrane of cellulose and then solidifying theresulting aqueous solution through drying. The domesticated silkwormsilk fibroin membrane can easily be biodegraded, as compared with thewild silkworm silk fibroin membrane. Therefore, a hardly biodegradablecomposite material comprising domesticated and wild silkworm silkfibroins may be obtained by increasing the content of the wild silkwormsilk fibroin present in the composite material.

When the biodegradable biopolymer material of the present invention isused while it is embedded in the living body, the material is ultimatelydecomposed into lower molecules, such as water and carbon dioxide, bythe action of enzymes present in the body, such as protease, and then isexcreted outside the body. The easily biodegradable domesticatedsilkworm silk fibroin membrane may be biodegraded within a relativelyshort period of time even when it is embedded in the body unlike thehardly biodegradable domesticated silkworm silk fibroin fibers.Therefore, the membrane can be used for temporarily helping therepairable damaged biological tissues in their healing or for thepreparation of a sustained release drugs, as discussed above. Theabsorptive material of the present invention may be used in a variety ofapplications, such as the suture of incised and/or wound portions,arrest of hemorrhage, bone fixation, a clue for tissue-regeneration anda means for preventing adhesion.

The biodegradable biopolymer material of the present invention isdigested and deteriorated through digestion with a protease. Therefore,it may also be used as a sustained release carrier for a usefulsubstance, such as a medicine or a physiologically active substance. Forinstance, when embedding, in the biodegradable biopolymer material, aproduct obtained by taking the useful substance in the biopolymermaterial or by fixing the useful substance to the biopolymer material,the useful substance is gradually released within the living body, whilethe biopolymer material is digested with enzymes present in the body.

Cellulose derivatives have been effectively utilized in various fields,such as food additives, cosmetics, additives for medicines andmedicines, such as an antithrombotic agent. Therefore, the compositematerial consisting of domesticated silkworm silk fibroin and cellulosemay be used in fields identical to those listed above in connection withcellulose. Moreover, the hybridization of domesticated silkworm silkfibroin with cellulose would permit the mechanical properties of thesilk fibroin, in particular, in its dried conditions. In addition, thehybridization of domesticated or wild silkworm silk fibroin with asecondary substance, such as cellulose, would permit the production of amaterial having improved moldability and transparency, as well as celladhesion properties, as compared with those observed for a membranesimply consisting of domesticated or wild silkworm silk fibroin.

The domesticated silkworm silk fibroin membrane may be relatively easilybiodegraded with protease. For this reason, if hybridizing thedomesticated silkworm silk fibroin with hardly biodegradable wildsilkworm silk fibroin, the resulting hybrid membrane would have acontrolled degree of biodegradation and may have an improvedfilm-forming ability and enhanced transparency.

The extent of biodegradation of the biodegradable biopolymer membraneaccording to the present invention can be controlled by a simpletreatment. Moreover, in a hybrid material, the biodegradable biopolymermoiety is firmly adhered to the surface of a secondary substance, thehybrid material is excellent in the wear resistance. Therefore, thesubstrate coated with a hybrid material is improved in the biologicalcell-growth properties on the surface thereof, as compared withsubstrate coated only with a protein. Accordingly, the hybrid materialis useful as a cell-growth substrate capable of being used in the fieldof biotechnology.

When immersing the biodegradable biopolymer material of the presentinvention in an aqueous solution containing antibacterial metal ions, alarge amount of such metal ions are adsorbed on the biopolymer material.Therefore, the biopolymer material carrying such metal ions adsorbedthereon is useful as an antibacterial fiber material. Moreover, whenimmersing the biopolymer material in waste water, it can adsorb metalions present in the waste water. Accordingly, the biopolymer material isalso effective as a fibrous material for adsorbing metal ions in wastewater.

The biodegradable biopolymer material of the present invention possesseswater-absorbing properties, which make the material applicable as awater-absorbable resin used in, for instance, disposable hygienic goodsand household goods, water cut-off agents, soil conditioners, dewinginhibitors, and water-retention agents for agriculture and horticulture.The present invention would also permit the supply of a water-absorbingmaterial having such biodegradability for a low price without requiringany complicated steps. For this reason, the material of the presentinvention can be applied to any fields of applications identical tothose for the conventionally known water-absorbing resins. For instance,the material of the present invention can be used in a wide variety offields, such as hygiene (typically use as a diaper and a sanitary good),medical services (for instance, use in cataplasms), civil engineeringand architecture (for instance, use as an agent for coagulating sludge),foods, industries, and agriculture and horticulture (for instance, useas a soil conditioner and a water-retention agent).

1. A biodegradable biopolymer material comprising a composite materialcomprising silk fibroin from wild silkworm and keratin from wool,wherein the keratin from wool is reduced keratin or S-carboxymethylkeratin.
 2. The biodegradable biopolymer material of claim 1, whereinthe biodegradable biopolymer material has a shape that is selected fromthe group consisting of sheet-like, membrane-like, powdery, bead-like,gel-like, fibrous, tubular, and hollow thread-like.
 3. A metalion-adsorbing material comprising a biodegradable biopolymer materialthat comprises: a composite material comprising silk fibroin from wildsilkworm and keratin from wool, wherein the keratin from wool is reducedkeratin or S-carboxymethyl keratin.
 4. The metal ion-adsorbing materialas set forth in claim 3, wherein the metal ions are antibacterial metalions or metal ions present in waste water.
 5. A sustained releasecarrier for a useful substance comprising a biodegradable biopolymermaterial that comprises: a composite material comprising silk fibroinfrom wild silkworm and keratin from wool, wherein the keratin from woolis reduced keratin or S-carboxymethyl keratin.
 6. The sustained releasecarrier for a useful substance as set forth in claim 5, wherein thebiodegradable biopolymer material is a porous substance.
 7. A substratefor growth of biological cells comprising a composite materialcomprising silk fibroin from wild silkworm and keratin from wool,wherein the substrate is used for the growth of biological cells, andwherein the keratin from wool is reduced keratin or S-carboxymethylkeratin.
 8. A method for the preparation of a membrane-likebiodegradable polymer material comprising silk fibroin from a wildsilkworm and keratin from wool, wherein the keratin is reduced keratinor S-carboxymethyl keratin, comprising the steps of: (a) applying, ontothe surface of a substrate, an aqueous mixed solution comprising anaqueous solution of silk fibroin from a wild silkworm and an aqueoussolution of keratin from wool, wherein the keratin from wool is reducedkeratin or S-carboxymethyl keratin, and (b) cast drying the appliedsolution to form the membrane-like biodegradable biopolymer material,wherein the aqueous mixed solution is prepared by uniformly admixing theconstituents of the aqueous mixed solution by stirring them such thatthey do not undergo any gelation, precipitation and/or coagulationreaction.
 9. A method for the preparation of a powdery biodegradablebiopolymer material comprising silk fibroin from a wild silkworm andkeratin from wool, wherein the keratin is reduced keratin orS-carboxymethyl keratin, comprising the steps of: (a) freezing anaqueous mixed solution comprising an aqueous solution of silk fibroinfrom wild silkworm and an aqueous solution of keratin from wool, whereinthe keratin from wool is reduced keratin or S-carboxymethyl keratin, andwherein the aqueous mixed solution is prepared by uniformly admixing theconstituents of the mixed aqueous solution by stirring them such thatthey do not undergo any gelation, precipitation and/or coagulationreaction, and (b) drying the frozen aqueous solution under a reducedpressure to form the powdery biodegradable biopolymer material.
 10. Amethod for the preparation of a gel-like biodegradable biopolymermaterial comprising silk fibroin from a wild silkworm and keratin fromwool, wherein the keratin is reduced keratin or S-carboxymethyl keratin,comprising the steps of: (a) adjusting the pH value of an aqueous mixedsolution comprising an aqueous solution of silk fibroin from wildsilkworm and an aqueous solution of keratin from wool, wherein thekeratin is reduced keratin or S-carboxymethyl keratin, to a pH levelwithin the acidic pH range, and (b) entirely coagulating the aqueousmixed solution to give the gel-like biodegradable biopolymer material.11. A method for the preparation of a porous body, comprising the stepof subjecting the gel-like biodegradable biopolymer material preparedaccording to the method set forth in claim 10 to lyophilization toobtain the porous body.
 12. The method as set forth in claim 8, whereinthe concentrations of the aqueous solution of the silk fibroin from wildsilkworm and the aqueous solution of the keratin from wool used in thepreparation of the mixed aqueous solution range from 0.1 to 5% w/v,respectively.
 13. The method as set forth in claim 9, wherein theconcentrations of the aqueous solution of the silk fibroin from wildsilkworm and the aqueous solution of the keratin from wool used in thepreparation of the mixed aqueous solution range from 0.1 to 5% w/v,respectively.
 14. The method as set forth in claim 10, wherein theconcentrations of the aqueous solution of the silk fibroin from wildsilkworm and the aqueous solution of the keratin from wool used in thepreparation of the mixed aqueous solution range from 0.1 to 5% w/v,respectively.
 15. The method as set forth in claim 11, wherein theconcentrations of the aqueous solution of the silk fibroin from wildsilkworm and the aqueous solution of the keratin from wool used in thepreparation of the mixed aqueous solution range from 0.1 to 5% w/v,respectively.